Assuming you are familiar with the ins-and-outs of Avernum's scripting language..
You could (and this is a somewhat vile solution) check the amount of characters in group 0 (which is the group that contains the PCs).
If it is greater than 2 (or one, if you want a singleton), then popup a window telling them to delete some characters and then end the scenario or block the space until they do.
If it is less than 3 (2 for singleton), then do nothing.
This is quite simple to do via a special node or terrain script. I have some code which does this (though mine inflicts a Dread Curse on players rather than making them delete their party members. It's easily adapted to do either.). The benefit of this is that you do not use pre-fabricated characters, and therefore it allows the player much more freedom in character creation.
By the way, the split party calls only work in the current town, as far as I recall (it was that way in Blades of Exile, at least). Therefore, it would not last for the duration of an entire scenario, unless the scenario consisted only of one town, of course.
A little warning: If you do this, make sure you do not have any towns in which you can create characters, as then they could simply create a new party member after they passed the special node. Of course, some clever players may come up with ways to bypass this limitation, but no technique is flawless.
Also note that this forces their characters to be deleted. It doesn't leave them in Limbo. All of their items will/should be dropped on the ground when they are deleted, but the characters themselves can not be recovered at the end of the scenario.
Players who have more characters than the set limit would have to make a new save file, or they would lose their party members eternally. They could not take their party into other scenarios once they completed yours, as they would not have their full party, and all the experience gained in your scenario would be for naught if they use their other party (the one with all their members) in other scenarios.