I find it hard to say at this point. To me, the most likely options seem to be Palutena, a new Fire Emblem character, or Takamaru. Mostly due to anniversary stuff, but there are a few things that make these candidates stand out to me.
Palutena: As others have pointed out, we've been getting a fairly large amount of Kid Icarus related material revealed as of late. Perhaps it's building up to something?
Fire Emblem Newcomer: I guess just anniversary stuff here. However, if Ike were to be revealed sometime before the Direct, I would say that the chances of a Fire Emblem newcomer for the Direct would skyrocket.
Takamaru: Well, they probably have some kind of retro character in the game, and they'd have to be revealed at some point. Probably not the best hype inducers, so a retro would probably be better suited to a Direct than E3. Could be wrong about that though... Anyway, if Takamaru is in the game, I think he'd be a good followup reveal to Little Mac, with him appealing to the Japanese fanbase after the western fanbase got a very special treat.
EDIT: Oh yeah, and I would think that, if there are any brand new third party characters to be revealed still, they would most likely be saved for E3. That's the best time to reveal such a big thing in the game, I think.