Since TCR-mediated stimulation induces T cells to become sensitive to Fas-mediated activation-induced cell death (Fas-AICD), we examined whether anergized and CD28-costimulated T cell clones were equally sensitive to Fas-AICD. Here, we show that TCR signal in the presence or absence of CD28 costimulation induced equivalent expression of Fas and Fas ligand. Although anergized cells expressed Fas and Fas ligand, they were resistant to Fas-AICD. Induction of anergy resulted in up-regulation and persistent expression of moderate amounts of bcl-xL and bax and absence of induction of bad. In contrast, CD28-costimulated cells that also expressed Fas and Fas ligand were initially resistant to Fas-AICD but became susceptible after 72 h of culture. Although Fas-mediated apoptosis was the major mechanism of AICD, the IL-1beta-converting enzyme-like protease inhibitor zVAD-FMK totally abrogated DNA fragmentation but not cell death, suggesting that additional Fas-independent apoptotic mechanisms were also operative. Resistance to apoptotic cell death was temporally associated with a dramatic increase of bcl-xL and the presence of bcl-xL:bax heterodimers. Subsequent sensitivity to AICD was associated with down-regulation of bcl-xL, induction of bad, and the displacement of bax from bcl-xL:bax heterodimers. Although induced following CD28 costimulation, bcl-2 did not protect against AICD. Therefore, besides its role in promotion of viability, prevention of anergy, and clonal expansion, CD28 costimulation also has a central role in the induction of subsequent AICD by up-regulating apoptotic mediators.