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.