TGF-β is a key immunoregulatory cytokine which supports self-tolerance by signaling to T cells. In this report, we show a crucial role for TGF-β signaling to T cells in enabling the long-term acceptance of allografts, whether natural or induced therapeutically by coreceptor and costimulation blockade. The requirement for TGF-β appears most pronounced during the initial exposure to alloantigens. We demonstrate the ability of TGF-β to direct the development in vitro of regulatory cells that suppress graft rejection in vivo. Such suppression was not affected by anti-TGF-β treatment of the recipient mice. Despite this, TGF-β may still have a role in CD4+ cell-mediated suppression of antiallograft responses in vivo, since its neutralization can, in some cases, abrogate suppression. These results show that TGF-β signaling to T cells is dispensable for mounting destructive responses against skin allografts while appearing to be an essential intermediary in establishing long-term tolerance.