Antigen-specificity is a hallmark of adaptive T cell-mediated immune responses. CD4⁺CD25⁺FOXP3⁺ regulatory T cells (T_R) also require activation through the T cell receptor for function. Although these cells require antigen-specific activation, they are generally able to suppress bystander T cell responses once activated. This raises the possibility that antigen-specific T_R may be useful therapeutically by localizing generalized suppressive activity to tissues expressing select target antigens. Here, we demonstrate that T_R specific for particular peptide-MHC complexes can be generated from human CD4⁺CD25^– T cells in vitro and isolated by using HLA class II tetramers. Influenza hemagglutinin epitopes were used to generate hemagglutinin-specific T_R, which required cognate antigen for activation but which subsequently suppressed noncognate bystander T cell responses as well. These findings have implications for the generation of therapeutic regulatory T cells in disease, and also suggest an important mechanism by which T cells may be regulated at the site of inflammation.