Ligation of CD28 during Ag recognition plays an important role in the generation of effective T cell responses. However, its peripheral control of regulatory T cell function remains obscure. In this study, we show that naive wild-type or CD28−/− CD4+ CD25− T cells exposed to peptide in vivo develop regulatory activity that suppresses the response of adoptively transferred naive T cells to a subsequent immunogenic challenge. We find that although CD28 is engaged during the initial peptide-priming event and is essential to sustain T cell survival, it is not sufficient to prevent the dominance of regulatory T cell function. Immunization with adjuvant abrogates regulatory dominance, reducing overall Foxp3 expression in a CD28-dependent manner. We conclude that CD28 licenses active immunization by regulating Ag-induced immunoregulation.