The low number of natural regulatory T cells (nTregs) in the circulation specific for a particular Ag and concerns about the bystander suppressive capacity of expanded nTregs presents a major clinical challenge for nTreg-based therapeutic treatment of autoimmune diseases. In the current study, we demonstrate that naive CD4+CD25-Foxp3- T cells specific for the myelin proteolipid protein (PLP)139-151 peptide can be converted into CD25+Foxp3+ induced Treg cells (iTregs) when stimulated in the presence of TGF-beta, retinoic acid, and IL-2. These PLP139-151-specific iTregs (139-iTregs) have a phenotype similar to nTregs, but additionally express an intermediate level of CD62L and a high level of CD103. Upon transfer into SJL/J mice, 139-iTregs undergo Ag-driven proliferation and are effective at suppressing induction of experimental autoimmune encephalomyelitis induced by the cognate PLP139-151 peptide, but not PLP178-191 or a mixture of the two peptides. Furthermore, 139-iTregs inhibit delayed-type hypersensitivity responses to PLP139-151, but not PLP178-191, myelin oligodendrocyte glycoprotein (MOG)35-55, or OVA323-339 in mice primed with a mixture of PLP139-151 and the other respective peptides. Additionally, 139-iTregs suppress the proliferation and activation of PLP139-151-, but not MOG35-55-specific CD4+ T cells in SJL/B6 F1 mice primed with a combination of PLP139-151 and MOG35-55. These findings suggest that Ag-specific iTregs are amplified in vivo when exposed to cognate Ag under inflammatory conditions, and these activated iTregs suppress CD4+ responder T cells in an Ag-specific manner.