During Spring Training if you remove a player for the 40 Man and he has options left, you'll either need to assign him to AAA/AA/Class A or if he is out of options, he'll need to clear Waivers before he is outrighted to the Minors.
Again, the easier option here is to just remove the players before you start your Franchise, that way they are already in minors and you can just add them to the 40 Man after Spring Training without burning through that players options moving him back and forth.