Natural T regulatory cells (nTreg) are an important subpopulation of CD4+ T cells generated in thymus, and essential in preventing autoimmune diseases. However, the role of MHC-class-II-mediated TCR signaling in nTreg development remains unclear, since nTreg still presents in MHC-class-II gene knock-out mice. We examined nTreg development in Helper Deficient (HD) mice, in which the majority of MHC-class-II-restricted, CD4 lineage designated T cells is mis-directed to the CD8 lineage due to a mutated zbtb7b/ThPOK, a key regulator of CD4+ cell development, gene. In HD, both percentage and number of conventional CD4+ T cells were reduced 10 folds, compared to wild-type littermates, but the total T cell number remained unchanged since >90% of MHC-class-II restricted CD4 T cells were mis-directed to develop as CD8+ cells. Unexpectedly, the percentage of FoxP3+ cells within the residual CD4+ T cells in HD was increased to >50% in spleen and lymph nodes, compared to 5–10% of CD4+ T cells in control mice, but the absolute numbers of FoxP3+CD4+ T cells was decreased by >50% in HD. In contrast, FoxP3+CD8+ cells, both percentage and numbers, were not significantly changed in HD. Similarly, there was no increase of re-directed FoxP3+CD8+ T cells in Beta-2-microglobulin KO x HD double mutants. Furthermore, after crossing HD with a FoxP3-EGFP reporter line, >90% EGFP+ T cells were CD4+ cells, with 1% detected EGFP+CD8+ cells. Functionally, the CD4+EGFP+ T cells isolated from HD inhibited TCR-triggered IL-2 production and CD69 up-regulation of CD4+EGFP− T cells. In summary, we demonstrated that, in contrast to MHC-class-II-restricted, conventional CD4 designated T cells are mis-directed to CD8 lineage, there is no re-directed CD8 nTreg developed in HD. Our data indicates that either commitment of MHC-class-II-restricted nTreg is earlier than conventional MHC-class-II-restricted CD4+ cells or selection/expansion of MHC-class-II-restricted nTreg is not supported in CD8+ T cells.