Injection of Ag into the thymus of adult animals induces specific systemic tolerance. The mechanisms of acquired thymic tolerance include anergy and the deletion of Ag-specific T cells. Here, we report that anergy to nominal Ag induced via acquired thymic tolerance requires CTL-associated Ag 4 (CTLA4) engagement. The role of CTLA4 in the induction and maintenance of tolerance was then investigated in the murine experimental autoimmune encephalomyelitis model. CTLA4 blockade abrogated the induction but not the maintenance phase of acquired thymic tolerance induced by intrathymic injection of myelin Ags. In addition, CTLA4 blockade had a restricted window of action after priming with Ag, which is consistent with the expression patterns of CTLA4 in vivo. We conclude that: 1) the induction of acquired thymic tolerance requires signaling through CTLA4 and 2) CTLA4 does not appear to be required for the maintenance of acquired thymic tolerance. This is the first report documenting the role of a CTLA4 negative signaling pathway in the induction of tolerance in an autoimmune disease model.