Ascended Meme: Issue #13 ends with Twilight, Pinkie, Rainbow, and Rarity facing against a giant ghost crab. "Rarity fighting a giant crab" has been a meme in the fanbase for some time, its origins itself pretty much out of the blue. (Someone asked to have artwork of Rarity fighting a crab, and these simply haven't stopped coming).
Blooper: Andy Price seems to have started a habit of forgetting pegasus wings. Funny enough this is a common animation error in the show. Honest mistakes or accuracy to the source material? You decide.
First issue, if you think "She'll get rid of them right quick!" is an odd thing for Rainbow Dash to say, this is because Heather Breckel accidentally gave Applejack RD's color scheme (the panel only shows a little bit of her face so it was probably an easy mistake).
Fandom Nod: In issue #8, Pinkie uses the term "pegasister" to refer to her friends. The fandom has used "pegasisters" to some degree to describe the female adult fans akin to the word "brony".
Promoted Bronies/Running the Asylum: Both Andy Price and Katie Cook had plenty of G4 fanart on their deviantART pages before the series was announced, and Katie has said she was a fan of the original G1 series. This leads to such things as Derpy being featured prominently and being fond of muffins, “flank” being used as a synonym for “butt,” etc.
Amy Mebberson, Heather Nuhfer, and Heather Breckel were also all big fans before working on the comic. Pretty much everyone involved is a brony/pegasister!
Andy Price originally wanted everything to be hand-done, including the interior colors, similar to how his covers are done. That would have taken far too much time, however, so the interiors are digitally colored.
Thom Zahler (creator of Love and Capes and also did the Twilight Sparkle spotlight issue) seriously pitched My Little Pony for IDW's Mars Attacks!Cross Through event. The plot would involve Princess Celestia casting a spell to disable the Martians' weapons, causing them to find hilarious new ways to attack ponies only to fail and eventually learn the magic of friendship. The Martians would return to their own dimenision to spread love & tolerance, only to confuse their fellow Martians that didn't visit Equestria. Alas, the Cross Through came and went, and no ponies...