Thymus‐derived CD4⁺CD25⁺ regulatory T (T_r) cells play a critical role in suppressing aberrant responses to self in vivo. The factors that influence a CD4⁺ T cell's decision to commit to an immunoregulatory T_r cell lineage are currently unknown. In the present study, we found that in mice, abundantly expressing a few or one peptide(s) bound to MHC class II molecules, a large portion of conventional CD4⁺ T cells could be biased towards the commitment to a T_r lineage by reducing the threshold required for thymocyte activation. This occurred in the presence of either an antisense glucocorticoid receptor transgene or a pharmacological inhibitor of glucocorticoid synthesis. These results demonstrate a novel in vivo pathway for the generation of T_r cells, and raise the possibility that therapeutic enhancement of the T_r cell repertoire through pharmacological manipulation of TCR signaling thresholds may provide a feasible means of ameliorating autoimmunity.