When Fluttershy is first introduced in the series, she barely manages to tell Twilight Sparkle her own name on account of her timidity, whispering so quietly that Twilight cannot hear her, and hiding her face behind her mane. However, she immediately becomes enthusiastic when she sees Spike, imploring the baby dragon to hold a conversation with her, which he ends up doing, and follows him and Twilight all the way to the library. Her shy nature contrasts with Rainbow Dash's abrasiveness, most notably in Dragonshy, and to a lesser extent in Sonic Rainboom. In season one, Fluttershy's inability to speak up often annoys Rainbow Dash, causing Rainbow to express her aggravation more than once. One instance is when Fluttershy cheers as loudly as she can for Rainbow Dash at the beginning of Sonic Rainboom, yet much to the latter's exasperation, can't manage more than a whisper. Season two shows them having grown closer to each other as friends despite their differences.
She is afraid of dragons (with the stated exception of baby dragons such as Spike), and is hesitant to continue forward when she and the others embark on an expedition to deal with a sleeping dragon in Dragonshy, relying heavily on her friends' encouragement. In the end, Fluttershy finds the courage to speak to the dragon when it begins harassing her friends, going so far as to raise her voice in anger and stand on its nose to look it in the eye as she lectures it. In Stare Master, she tames a wild cockatrice in a similar manner, becoming angry with it when it scares the Cutie Mark Crusaders; however, in this instance, the Stare is employed.
She is reduced to tears by Gilda in Griffon the Brush Off when the griffon roars in her face. Afraid to be in the same room as Gilda during Pinkie Pie's party, Fluttershy attempts to voice her discomfort to Pinkie, who obliviously waves off her distress.
In Luna Eclipsed, Fluttershy sequesters herself at home in her cottage during Nightmare Night, and refuses to answer the door until she learns that her visitor is Twilight. She is then forcibly brought out by Twilight to give Princess Luna a lesson in how to speak softly, with the Princess's "Royal Canterlot Voice" terrifying her.
In Putting Your Hoof Down, Fluttershy becomes "assertive" after attending a seminar from Iron Will. However, when she over-asserts herself, (e.g. going berserk when a mail carrier delivers her the wrong mail), Rarity and Pinkie attempt to intervene. In the end, she realizes her mistake after driving her friends away in tears, and with the help of her animal friends, locks herself in her cottage to prevent herself from causing anyone more harm. When Iron Will visits shortly afterwards to collect the fee for his seminar, Fluttershy uncharacteristically politely refuses him, eliciting shock from the onlooking Rarity and Pinkie Pie. She explains that she is evoking Iron Will's earlier guarantee that if a customer was not satisfied with his services, they would pay nothing; as she is not satisfied, she will not pay him. Iron Will relents and leaves, and her friends congratulate her on her new-found confidence.
In Dragon Quest, she again displays her fear of dragons. Fluttershy pointedly refuses to watch the Dragon Migration with her friends, despite Twilight's coaxing. It culminates in Fluttershy pushing past Rainbow Dash and jumping out the window, fleeing the scene before any of them could follow her. In Hurricane Fluttershy, she tries to evade her responsibilities as a Pegasus to help create a tornado to move water to Cloudsdale from Ponyville's reservoir. After some insistence from Rainbow Dash, she reveals that she is afraid of humiliating herself by performing badly as a flyer in front of all the other Pegasi. She becomes even more discouraged after the anemometer (wing-power gauging machine) gives her wing-power as only 0.5. Her animals' motivation causes her to engage in a period of training and exercise, but her second attempt only puts her at 2.3, further diminishing her hopes for improvement. In the end, Twilight convinces her to help when the other Pegasi's wingpower proves not to be enough to create the tornado. With a sudden burst of determination, she manages to surpass all the other Pegasi in flying speed, completing the 800 wingpower minimum to create the tornado and resulting in the water successfully reaching Cloudsdale. In Castle Mane-ia she displays fear at the possibility of running into star spiders. In Issue #2 of the IDW comic, The Return of Queen Chrysalis, however, she feels quite differently, showing an interest in spiders, and disappointment that "Fluffy" the giant tarantula turned out to be mean, saying that most spiders are docile.
When the ponies venture into the Everfree Forest seeking the Elements of Harmony, she is the only pony who does not engage in a fight with a Manticore when it attacks them, and relieves a thorn embedded in its front paw. She later reveals to Twilight that she did not know about the thorn, but she showed consideration to the fierce Manticore regardless. This kind act entitles her to being the wielder of the element of kindness, as well as reveals her faithful and considerate handling of all types of animals. After humbling the dragon in Dragonshy and making it cry, she immediately consoles it and advises it to look for a more suitable place to sleep, successfully driving it away. In The Return of Harmony Part 1, she is the only pony who isn't easily affected by Discord's manipulation, prompting him to forcefully hypnotize her into her polar opposite (in her case, cruelty). In this state, she appears to take pleasure in causing others grief and distress, such as trampling the flowers in front of the library, dumping water on Twilight Sparkle and Spike and snatching the Elements of Harmony from Twilight. When Twilight Sparkle sets out to reverse Discord's effect on her friends using a "memory spell", Twilight and Applejack are forced to hogtie her and pin her to the floor, with Applejack standing on her tail to prevent her from flying away. Upon returning to normal, a horrified Fluttershy initially believes her corrupted state to be a "horrible dream".
In Keep Calm and Flutter On, she is the only one of the main six who believes Discord can be redeemed. She shows him genuine kindness and friendship, eventually leading him to change his ways.
Despite being soft-spoken and extremely polite most of the time, Fluttershy has shown a more aggressive side on rare occasions. Most of the time, Fluttershy can withstand almost anything that will make other ponies explode in anger. But even a shy Pegasus like Fluttershy has her own limits. In The Best Night Ever, Fluttershy slowly becomes frustrated when the animals living in the Canterlot castle gardens avoid her. Instead of continuing her kind and calm ways to earn the animal's trust, she decides to make them love her by setting animal traps, hoping to trap them in a cage. Eventually, the shy Pegasus explodes, yelling and screaming, wanting the animals to love her.
Fluttershy becomes angry when reminded that Discord will win if she is not able to catch Rainbow Dash in The Return of Harmony Part 2 (referring to Discord as a 'big dumb meanie'). This is one instance where Fluttershy doesn't explode in anger, but expresses her anger in words.
In Putting Your Hoof Down, Fluttershy is too much of a "doormat", where a pony can easily take advantage of the situation to their own benefit. For example, if a pony purposely cuts in front of line ahead of Fluttershy, she apologizes, saying that the pony can go first. Pinkie Pie and Rarity say that Fluttershy should stand up for herself so that she isn't pushed around. After receiving motivational advice from Iron Will, a manly minotaur that helps ponies like Fluttershy, she becomes progressively more violent and aggressive, to the point of reducing Pinkie Pie and Rarity to tears with her insults. However, she changes her ways by the end of the episode.