Antigen cross-presentation by dendritic cells is crucial for priming cytotoxic CD8(+) T cells to invading pathogens and tumour antigens, as well as mediating peripheral tolerance to self-antigens. The protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor (TCR) signalling and tunes CD8(+) T cell responses in vivo. In this study we have examined the role of PTPN2 in the maintenance of peripheral tolerance after the cross-presentation of pancreatic β-cell antigens. The transfer of OVA-specific OT-I CD8(+) T cells (C57BL/6) into RIP-mOVA recipients expressing OVA in pancreatic β-cells only results in islet destruction when OVA-specific CD4(+) T cells are co-transferred. Herein we report that PTPN2-deficient OT-I CD8(+) T cells transferred into RIP-mOVA recipients acquire CTL activity and result in β cell destruction and the development of diabetes in the absence of CD4(+) help. These studies identify PTPN2 as a critical mediator of peripheral T cell tolerance limiting CD8(+) T cell responses after the cross-presentation of self-antigens. Our findings reveal a mechanism by which PTPN2 SNPs might convert a tolerogenic CD8(+) T cell response into one capable of causing the destruction of pancreatic β-cells. Moreover, our results provide insight into potential approaches for enhancing T cell-mediated immunity and/or T cell adoptive tumour immunotherapy.