OX40 is a member of the TNFR superfamily and has potent T cell costimulatory activities. OX40 also inhibits the induction of Foxp3+ regulatory T cells (Tregs) from T effector cells, but the precise mechanism of such inhibition remains unknown. In the present study, we found that CD4+ T effector cells from OX40 ligand-transgenic (OX40Ltg) mice are highly resistant to TGF-β mediated induction of Foxp3+ Tregs, whereas wild-type B6 and OX40 knockout CD4+ T effector cells can be readily converted to Foxp3+ T cells. We also found that CD4+ T effector cells from OX40Ltg mice are heterogeneous and contain a large population of CD44 high CD62L− memory T cells. Analysis of purified OX40Ltg naive and memory CD4+ T effector cells showed that memory CD4+ T cells not only resist the induction of Foxp3+ T cells but also actively suppress the conversion of naive CD4+ T effector cells to Foxp3+ Tregs. This suppression is mediated by the production of IFN-γ by memory T cells but not by cell-cell contact and also involves the induction of T-bet. Importantly, memory CD4+ T cells have a broad impact on the induction of Foxp3+ Tregs regardless of their origins and Ag specificities. Our data suggest that one of the mechanisms by which OX40 inhibits the induction of Foxp3+ Tregs is by inducing memory T cells in vivo. This finding may have important clinical implications in tolerance induction to transplanted tissues.