Alphabet Lore has plenty of serious moments that are Played for Laughs.
- When F first finds D, he comically knocks him out by hitting him a few times in rapid succession, in other words, D was F'D. When he finds E, he then proceeds to knock him out with D's body.
- By the time O begins his superhero transformation, F is just tired of the whole process. As such, he sits down on I as O's transformation goes on offscreen, involving several explosions, before O gives the tiniest of taps to F's snout before rolling away.
- Y's Skyward Scream when reacting to X's dead body is the most unexpected uses of Fun with Homophones, to the point where it's just absolutely hilarious.
- Lowercase F using A, R, and T to spell "fart" as revenge for being up N.
- 1 initially looks like a terrifying Big Bad, but after seeing his Muppet style talk, it's made clear that he's just a Cloud Cuckoolander who takes his job seriously.
- Q trying to save M. The slow motion and his scream parody a Big "NO!" all set up for the Bait-and-Switch with M being constantly bruised with 2's infinite teeth.
- A and E's rivalry on who is deemed more important in the AEIOU team, hogging each other's spotlight for more attention.
- G and P rapidly kissing their baby, then the baby gets unintentionally taken away by V, A and N as a van as they pass by.
