CD4⁺25⁺ T cells are a unique population of immunoregulatory T cells which are critical for the prevention of autoimmunity. To address the thymic selection of these cells we have used two models of attenuated thymic deletion. In K14-A_β^b mice, major histocompatibility complex (MHC) class II I-A^b expression is limited to thymic cortical epithelium and deletion by hematopoietic antigen-presenting cells does not occur. In H2-DMα–deficient mice, MHC class II molecules contain a limited array of self-peptides resulting in inefficient clonal deletion. We find that CD4⁺25⁺ T cells are present in the thymus and periphery of K14-A_β^b and H2-DMα–deficient mice and, like their wild-type counterparts, suppress the proliferation of cocultured CD4⁺25⁻ effector T cells. In contrast, CD4⁺25⁺ T cells from MHC class II–deficient mice do not suppress responder CD4⁺ T cells in vitro or in vivo. Thus, development of regulatory CD4⁺25⁺ T cells is dependent on MHC class II-positive thymic cortical epithelium. Furthermore, analysis of the specificities of CD4⁺25⁺ T cells in K14-A_β^b and H2-DMα–deficient mice suggests that a subset of CD4⁺25⁺ T cells is subject to negative selection on hematopoietic antigen-presenting cells.