Itoje, who had sustained medial ligament damage during England's 32-20 win over holders Ireland in their opening game, suffered a setback in his bid to recover before Saturday's win against Italy when he limped out of training last week.
"Maro Itoje (Saracens) has returned to his club to continue to rehabilitation," the RFU said in a statement.
England coach Eddie Jones called up 31 players to prepare for their final match against Scotland at Twickenham, including uncapped quartet Ben Earl, Elliott Stooke, Marcus Smith and Ollie Thorley.
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Meanwhile, Scotland will be without back-three trio Tommy Seymour (rib), Blair Kinghorn (ankle) and Stuart Hogg (shoulder) due to injuries, Scottish Rugby said in a statement.
Seymour and Kinghorn were injured in Saturday's 18-11 defeat by Wales and the England clash comes too soon for them.
Hogg is recovering from a shoulder injury sustained in the 22-13 loss to holders Ireland in the second round last month.
Head coach Gregor Townsend called up Glasgow Warriors' Ruaridh Jackson and uncapped Kyle Steyn and released Adam Ashe, Tim Swinson, James Johnstone and Luke Crosbie.
England, who have beaten Ireland, France and Italy to sit second with 15 points, can still win the championship if they overcome Scotland and table-toppers Wales lose to Ireland.
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