Flood, 28, with 60 caps to his name, has said he will leave Leicester and though his destination is yet to be confirmed, Toulouse look the strong favourites.
That means he has fallen foul of the Rugby Football Union's (RFU) policy of not picking overseas-based players apart from in "exceptional circumstances".
Flood is replaced in the squad by uncapped George Ford, who joins Freddie Burns and Owen Farrell as coach Stuart Lancaster's flyhalf options.
"I have met Toby Flood several times and told him I really wanted him to stay at Leicester and play for England," Lancaster said in an RFU statement.
"He has made a lifestyle choice and we respect that but given the RFU's policy of not selecting players based overseas save for exceptional circumstances it was important that we allow others such as George Ford to progress."
Flood made his England debut in 2006 but, after finally nailing down the flyhalf berth, found himself behind Farrell in the pecking order once Lancaster became coach two years ago. Twenty six of his caps have come off the bench.
Ford, a former understudy of Flood at Leicester, has blossomed since moving to Bath this season and was promoted from the second-string Saxons squad.
Ford, 20, won the world junior World Player of the Year in 2011, the only northern hemisphere player to have done so, and was chosen ahead of Danny Cipriani, who had put himself back in the frame with some encouraging performances for Bath.
Players outside the EPS squad can still be drafted in by Lancaster but, with the World Cup less than two years away, time is running out for anyone outside it to force their way into contention.
In addition to Ford, Lancaster called-up eight other players from the England Saxons to join a Six Nations championship training squad as injury replacements.
Luther Burrell, Tom Johnson, Jonny May, Matt Mullan, Jack Nowell, Ed Slater, Henry Thomas and Anthony Watson replace Manusamoa Tuilagi, Tom Croft, Marland Yarde, Alex Corbisiero, Christian Wade, Geoff Parling, David Wilson and Ben Foden.
England open their Six Nations campaign away to France on February 1 and also play New Zealand in a three-test series in June.
(Reporting by Mitch Phillips/Mike Collett, editing by Justin Palmer)
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