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). Funny enough, Rarity doesn't really contribute to the crab fight.note She does hold her own in a sword fight with Hoofbeard, so she's still awesome
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".
In Issue #18, when the mane 6 are attacked by the evil Celestia and Luna in the mirror universe, Rarity accuses Luna's choice of red and black colors for her armor sarcastically "original". In the fandom, there are a lot of fan's original character ponies using similar red and black (especially on alicorn characters), making the color scheme completely unoriginal and a joke that such colors are a part of Donut Steel (O Cs made with "Do Not Steal" warnings despite no one wanting to steal them).
Some of the gags, especially ones involving background ponies (see Wolverine Publicity below) or recreating popular moments from the show.
Commander Cocoon for the green armored advisor of Queen Chrysalis in that same issue.
Nightmarity and Night Rare Moon for Nightmare Rarity.
Jossed: One panel in Issue #11 has Shining and Twilight's parents helping Shining get ready for a recital at their home. On the wall are a few plaques and awards that refer to Twilight Velvet (the apparent name for their mother), including one that says "Daring Do". Many fans took this as Twilight Velvet was the award-winning writer of the Daring Do books, collaborating with one wave of blind bags that include Twilight Velvet that called out that she "loves to write about adventures!" This was all Jossed with the fourth season episode "Daring Don't" that established Daring was a real character in Equestria and wrote her own stories under the name "A.K. Yearling". However, this was also a case of What Could Have Been as it was planned in the comics to reveal Twilight's mother as the writer, but was forced to backtrack when "Daring Don't" was released, saying that the cartoon supersedes the comic in canon terms.
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”, several G1 references, 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!
Schedule Slip: Not to extreme extents, but each issue of the first Story Arc have had to be delayed from their original scheduled release date. The first issue due to demand for the comics far exceeding expectations, the second (with a bunch of other IDW comics) due to a union strike, and the last two due to customs inspections.note IDW comics are printed overseas
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 dimension 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...
Artist Brenda Hickey had original named Jewel as Coralita, and has a design similar to the ponies. Hasbro asked for the design to be made more in line with Under the Sparkling Sea, and changed her name to Jewel to avoid the existing name Coral from the book.
The cats in Issue #24 were going to have cutie marks on their foreheads and more direct coloring matches to the Mane 6 as to emphasize the connection more between them and the ponies; according to Hickey, Hasbro nixed this approach, but still allowed for enough of the Mane 6 coloring and other style to show through to make the joke obvious. One panel in the final version has Scootaloo feeling at her forehead as where the cutie marks would have been, which Hickey left in, feeling it would still be apparent what the connection was.