CD4+CD25+ regulatory T cells (Tregs) play an essential role in controlling autoimmunity and allograft rejection. Several ex vivo activation and expansion protocols have been developed to amplify cell numbers and suppressor function of murine and human Tregs. We demonstrate in this study that ex vivo activation and expansion of murine Tregs resulted in an enrichment of a CD4low/negCD25+ T cell population that was more than 20-fold more potent than expanded conventional Tregs in suppressing an in vitro CD4+CD25̄ T cell response to allo-Ag. The generation of CD4low/negCD25+ T cells was independent of the presence of Tregs in the culture, and suppressor function was acquired only after activation and expansion. CD4low/negCD25+ T cells expressed either an αβ or γδ TCR, had an activated phenotype, and did not express the transcription factor FoxP3. Despite expressing the cell surface Ags lymphocyte activation gene-3 (CD223) and CD103, neither was essential for suppressor cell function. Suppression by CD4low/negCD25+ T cells was prevented by a semipermeable membrane and was independent of IL-10 and TGF-β. In summary, we describe in this study CD4 low/negCD25+ FoxP3neg T cells with highly potent suppressor cell function derived from cultures of an enriched population of CD4+CD25+ T cells that may contribute to the suppressor activity of ex vivo expanded bone fide Tregs.