This study demonstrates that CD4⁺ T cells specific for an altered self‐antigen differentiate to T cells secreting transforming growth factor (TGF)‐β1. In this study, we utilized mice expressing an altered peptide ligand containing a single amino acid substitution of moth cytochrome c 88–103 peptide. In these mice, antigen‐specific T cells escaping thymic negative selection differentiated into T cells with an effector/memory phenotype, CD44^high, CD45RB^low, CD62L^– and CD25^intermediate. The expression of CD25 and high levels of CD44 was initiated in the thymus during the development from CD4⁺CD8⁺ to CD4⁺; a large proportion of maturing CD4⁺ thymocytes expressed both CD25 and high levels of CD44. Upon antigen stimulation, CD4⁺ T cells derived from these mice did not proliferate or secrete IL‐2, but secreted TGF‐β1. Neutralizing antibodies to TGF‐β1 reversed the impaired proliferative responses to the antigen, suggesting that TGF‐β1 secreted from these T cells negatively regulates T cell responses.