When Sweetie Belle asks the player to try out dialogue options, the only option is "Got it Squeaky Belle". Made even funnier by MissBunniSwan's voice crack immediately after that.
Pinkie Pie's Traitor Shot after the player meets Applejack and Apple Bloom. She's so close that she appears in front of the Letter Box.
When Rarity says she doesn't want to pull the cart, you can hear a faint "Hmm..." at the end of the line. That's not in the text box, meaning it was apparently ad-libbed.
This part from the scene where the player meets the CMC.
Apple Bloom: That's right, you're new in town. So ah guess you're tryin to earn up some bits. *Gasps* If you'd like ah could ask mah sister if she needs help down at the farm! Her and Big Macintosh are always super busy. Ah reckon they could use a helping hoof.
Scootaloo: Maybe that will get us our cutie marks. Cutie Mark Crusader job finders!
Applejack: Ah ain't ever seen a pony buck themselves onto their face before! Here, let me show ya one more time. Ya see, ya gotta get a good grip with yur front hooves, else yur gonna fall on yur face, as I reckon' y'all know. Haha.
The introduction of Spike.
Rarity: They're extremely rare. I got it as a gift, a special gift from my good friend Spike. It was sooo cute! He's such a sweetheart.