Homeostatic proliferation of T cells leads to the generation of effector/memory cells, which have the potential to cause harm to the host. The role of Tregs in the control of homeostatic proliferation is unclear. In this study we utilized mice that either harbor or lack Tregs as recipients of monoclonal or polyclonal T cells. We observed that while Tregs completely prevented cell division of T cells displaying low affinity for self ligands, they had a less marked, albeit significant, effect on cell cycle entry of T cells displaying higher affinity. The presence of Tregs resulted in a lower accumulation of T cells, enhanced apoptosis, and impaired differentiation to a cytokine-producing state. We conclude that Tregs play a major role in the control of homeostatic proliferation.