In vivo models have shown that tissue-restricted antigen may be captured by bone marrow–derived cells and cross-presented for the tolerization of CD8⁺ T cells. Although these studies have shown peripheral tolerization of CD8⁺ T cells, the mechanism of antigen transfer and the nature of the antigen-presenting cell (APC) remain undefined. We report here the establishment of an in vitro system for the study of cross-tolerance and show that dendritic cells (DCs) phagocytose apoptotic cells and tolerize antigen-specific CD8⁺ T cells when cognate CD4⁺ T helper cells are absent. Using this system, we directly tested the “two-signal” hypothesis for the regulation of priming versus tolerance. We found that the same CD83⁺ myeloid-derived DCs were required for both cross-priming and cross-tolerance. These data suggested that the current model for peripheral T cell tolerance, “signal 1 in the absence of signal 2”, requires refinement: the critical checkpoint is not DC maturation, but instead the presence of a third signal, which is active at the DC–CD4⁺ T cell interface.