Abstract Expression of the transcription factor FoxP3 is the hallmark of regulatory T cells that play a crucial role in dampening immune responses. A comparison of the development and phenotype of FoxP3+ T cells in relation to the expression of conventional MHC molecules facilitated the identification of several distinct lineages of naive and effector/memory populations of Foxp3+ T cells. One subpopulation of effector/memory Foxp3+ T cells develops in the thymic medulla, whereas the second is thymic independent. Both lineages display a distinct activated phenotype, undergo extensive steady-state proliferation, home to sites of acute inflammation, and are unique in their capacity to mediate Ag-nonspecific suppression of T cell activation directly ex vivo. Effector FoxP3+ T cells may act as a sentinel of tolerance, providing a first line of defense against potentially harmful responses by rapidly suppressing immunity to peripheral self-Ags.