Despite the impressive protection of B cell-deficient (muMT(-/-)) nonobese diabetic (NOD) mice from spontaneous diabetes, existence of mild pancreatic islet inflammation in these mice indicates that initial autoimmune targeting of beta cells has occurred. Furthermore, muMT(-/-) NOD mice are shown to harbor a latent repertoire of diabetogenic T cells, as evidenced by their susceptibility to cyclophosphamide-induced diabetes. The quiescence of this pool of islet-reactive T cells may be a consequence of impaired activation of T lymphocytes in B cell-deficient NOD mice. In this regard, in vitro anti-CD3-mediated stimulation demonstrates impaired activation of lymph node CD4 T cells in muMT(-/-) NOD mice as compared with that of wild-type counterparts, a deficiency that is correlated with an exaggerated CD4 T cell:APC ratio in lymph nodes of muMT(-/-) NOD mice. This feature points to an insufficient availability of APC costimulation on a per T cell basis, resulting in impaired CD4 T cell activation in lymph nodes of muMT(-/-) NOD mice. In accordance with these findings, an islet-reactive CD4 T cell clonotype undergoes suboptimal activation in pancreatic lymph nodes of muMT(-/-) NOD recipients. Overall, the present study indicates that B cells in the pancreatic lymph node microenvironment are critical in overcoming a checkpoint involving the provision of optimal costimulation to islet-reactive NOD CD4 T cells.