Cross-presentation by CD8(+) conventional dendritic cells (cDCs) is involved in the maintenance of peripheral tolerance and this process is termed cross-tolerance. Previous reports showed that non-obese diabetic (NOD) mice have reduced number of splenic CD8(+) cDCs compared with non-diabetic strains, and that the administration of Flt3L to enhance DC development resulted in reduced diabetes incidence. As CD8(+) cDCs are the most efficient antigen cross-presenting cells, it was assumed that reduced cross-presentation by non-activated, tolerogenic CD8(+) cDC predisposes to autoimmune diabetogenesis. Here we show for the first time that indeed NOD mice have a defect in autoantigen cross-presentation capacity. First, we showed that NOD CD8(+) cDCs were less sensitive to iatrogenic cytochrome c, which had previously been shown to selectively deplete CD8(+) cDCs that functionally cross-present. Second, we found that proliferation of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD8(+) T cells was impaired in NOD compared with non-obese diabetes resistant mice after immunization with cell associated recombinant fusion protein containing the cognate IGRP peptide. This study, therefore, suggests that the reduced number of CD8(+) cDCs in NOD mice, coupled with the reduced capacity to cross-present self-antigens, reduces the overall capacity to maintain peripheral tolerance in the spontaneous autoimmune type 1 diabetes mice.