Regulatory CD4+ CD25+ Foxp3+ T cells play a critical role in controlling autoimmunity and T cell homeostasis. However, their role in regulation of lymphopenia-induced proliferation (LIP), a potential mechanism for generation of autoaggressive T cells, has been poorly defined. Currently, two forms of LIP are recognized: spontaneous and homeostatic. Spontaneous LIP is characterized by fast, burst-like cell-cycle activity, and may allow effector T cell differentiation. Homeostatic LIP is characterized by slow and steady cell cycle activity and is not associated with the acquisition of an effector phenotype. In this study, we demonstrate that CD4+ CD25+ Foxp3+ T cells suppress the spontaneous, but not homeostatic, LIP of naive CD8 and CD4 T cells. However, selective inhibition of spontaneous LIP does not fully explain the tolerogenic role of Tregs in lymphopenia-associated autoimmunity. We show here that suppression of LIP in the lymphoid tissues is independent of Treg-derived IL-10. However, IL-10-deficient Tregs are partially defective in their ability to prevent colitis caused by adoptive transfer of CD4 T cells into RAG−/− mice. We propose that Tregs may inhibit emergence of effector T cells during the inductive phase of the immune response in the secondary lymphoid tissues by IL-10-independent mechanisms. In contrast, Treg-mediated inhibition of established effector T cells does require IL-10. Both Treg functions appear to be important in control of lymphopenia-associated autoimmunity.