FcR-binding “classical” anti-CD3 mAb is a potent immunosuppressive drug that alters CD4+ and CD8+ T cell function in vivo via anergy induction and programmed cell death (PCD). Anti-CD3-mediated PCD was Fas independent but was mediated by the mitochondria-initiated apoptosis that was abrogated in Bcl-x L-transgenic T cells. The PCD was more pronounced in CD28-deficient mice consistent with defective Bcl-x L up-regulation. Residual T cells isolated from anti-CD3-treated wild-type, CD28−/−, and Bcl-x L-transgenic mice were hyporesponsive. The hyporesponsiveness was more pronounced in CD28−/− and wild-type mice treated with anti-B7-2, suggesting that CD28 interaction with B7-2 regulates T cell responsiveness in anti-CD3-treated animals. Finally, anti-CD3 treatment led to indefinite cardiac allograft survival in wild-type but not Bcl-x L animals. Together these results implicate CD28/B7 signaling in the regulation of both anti-CD3-induced T cell depletion and hyporesponsiveness in vivo, but T cell depletion, not hyporesponsiveness, appears to be critical for anti-CD3 mAb-mediated long-term immune regulation.