Monoclonal anti-CD3 Abs have been used clinically for two decades to reverse steroid-resistant acute graft rejection. In autoimmune diabetes, short course treatment with FcR-nonbinding (FNB) anti-CD3 mAb in mice with recent onset of diabetes induces long-term disease remission. Induction of tolerogenic regulatory T cells (Tregs) has been implicated to be one of the mechanisms of action by FNB anti-CD3 mAb in these settings. In this study, we examined the effect of FNB anti-CD3 mAb treatment on the homeostasis of naive, effector, and regulatory T cells in vivo. Anti-CD3 treatment induced a transient systemic rise in the percentage but not absolute number of CD4(+)Foxp3(+) Tregs due to selective depletion of CD4(+)Foxp3(-) conventional T cells. T cell depletion induced by FNB anti-CD3 mAb was independent of the proapoptotic proteins Fas, caspase-3, and Bim and was not inhibited by overexpression of the anti-apoptotic protein, Bcl-2. Tregs were not preferentially expanded and we found no evidence of conversion of conventional T cells into Tregs, suggesting that the pre-existing Tregs are resistant to anti-CD3-induced cell death. Interestingly, expression of the transcription factor Helios, which is expressed by thymus-derived natural Tregs, was increased in Tregs after FNB anti-CD3 mAb treatment, suggesting that the anti-CD3 treatment can alter, and potentially stabilize, Treg function. Taken together, the results suggest that FNB anti-CD3 therapy promotes tolerance by restoring the balance between pathogenic and regulatory T cells.