It has recently been shown that CD4⁺CD25⁺ T cells are immunoregulatory T cells that prevent CD4⁺ T-cell–mediated organ-specific autoimmune diseases. In this study, the regulatory mechanism of CD4⁺CD25⁺ T-cell development were investigated using T-cell receptor (TCR) transgenic mice. It was found that CD4⁺CD25⁺ T cells preferentially expressed the endogenous TCRα chain in DO10⁺ TCR transgenic mice compared with CD4⁺CD25⁻ T cells. Moreover, it was found that CD4⁺CD25⁺ thymocytes were severely decreased in DO10⁺ TCR-α^−/− mice in positively selecting and negatively selecting backgrounds, whereas CD4⁺CD25⁻ thymocytes efficiently developed by transgenic TCR in DO10⁺ TCR-α^−/− mice in positively selecting backgrounds, indicating that the appropriate affinity of TCR to major histocompatibility complex (MHC) for the development of CD4⁺CD25⁺ thymocytes is different from that of CD4⁺CD25⁻ thymocytes and that a certain TCR–MHC affinity is required for the development of CD4⁺CD25⁺ thymocytes. Finally, it was found that, in contrast to thymus, CD4⁺CD25⁺ T cells were readily detected in spleen of DO10⁺TCR-α^−/− mice in positively selecting backgrounds and that splenic CD4⁺CD25⁺ T cells, but not CD4⁺CD25⁺ thymocytes, were significantly decreased in B-cell–deficient mice, suggesting that B cells may control the peripheral pool of CD4⁺CD25⁺ T cells. Together, these results indicate that the development of CD4⁺CD25⁺ T cells in thymus and the homeostasis of CD4⁺CD25⁺ T cells in periphery are regulated by distinct mechanisms.