Thymocytes differentiate into CD4(+) Foxp3(+) regulatory T cells (T(R)) upon interaction between their TCR and peptide-MHC II complexes locally expressed in the thymus. Conversion of naive CD4(+) T cells into T(R) can additionally take place in the periphery under noninflammatory conditions of Ag encounter. In this study, making use of TCR transgenic models naturally devoid of Foxp3(+) cells, we report de novo generation of T(R) upon a single footpad injection of Ag mixed with a classic proinflammatory adjuvant. Abrupt T(R) differentiation upon immunization occurred intrathymically and was essential for robust tolerance induction in a mouse model of spontaneous encephalomyelitis. This phenomenon could be attributed to a specific feature of thymocytes, which, in contrast to mature peripheral CD4(+) T cells, were insensitive to the inhibitory effects of IL-6 on the induction of Foxp3 expression. Our findings uncover a pathway for T(R) generation with major implications for immunity and tolerance induction.