Abstract T cell activation and tolerance are delicately regulated by costimulatory molecules. Although B and T lymphocyte attenuator (BTLA) has been shown as a negative regulator for T cell activation, its role in peripheral T cell tolerance induction in vivo has not been addressed. In this study, we generated a novel strain of BTLA-deficient mice and used three different models to characterize the function of BTLA in controlling T cell tolerance. In an oral tolerance model, BTLA-deficient mice were found resistant to the induction of T cell tolerance to an oral Ag. Moreover, compared with wild-type OT-II cells, BTLA−/− OT-II cells were less susceptible to tolerance induction by a high-dose OVA peptide administered i.v. Finally, BTLA−/− OT-I cells caused autoimmune diabetes in RIP-mOVA recipient mice. Our results thus demonstrate an important role for BTLA in the induction of peripheral tolerance of both CD4+ and CD8+ T cells in vivo.