Granzyme B (GZB) has been implicated as an effector mechanism in regulatory T cells (T reg) suppression. In a model of T reg-dependent graft tolerance, it is shown that GZB-deficient mice are unable to establish long-term tolerance. Moreover, mice overexpressing the inhibitor of GZB, serine protease inhibitor 6, are also resistant to tolerization to alloantigen. Graft survival was shorter in bone marrow-mixed chimeras reconstituted with GZB-deficient T reg as compared with wild-type T reg. Whereas there was no difference in graft survival in mixed chimeras reconstituted with wild-type, perforin-deficient, or Fas ligand-deficient T reg. Finally, data also show that if alloreactive effectors cannot express FoxP3 and be induced to convert in the presence of competent T reg, then graft tolerance is lost. Our data are the first in vivo data to implicate GZB expression by T reg in sustaining long-lived graft survival.