CD4+ CD25+ regulatory T cells (T reg) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on T reg has been shown to be a key player of regulating T reg-mediated suppression. A recent study reports that NF-κB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of T reg but not for its suppression capacity. In this report, we have shown that T reg from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive T reg proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type T reg. Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+ CD25+ CD62L low T reg both in thymus and periphery than their wild-type littermates. This CD62 low subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+ CD25+ regulatory T cells.