In addition to thymus-derived or natural T regulatory (nT reg) cells, a second subset of induced T regulatory (iT reg) cells arises de novo from conventional CD4+ T cells in the periphery. The function of iT reg cells in tolerance was examined in a CD45RB high CD4+ T cell transfer model of colitis. In situ-generated iT reg cells were similar to nT reg cells in their capacity to suppress T cell proliferation in vitro and their absence in vivo accelerated bowel disease. Treatment with nT reg cells resolved the colitis, but only when iT reg cells were also present. Although iT reg cells required Foxp3 for suppressive activity and phenotypic stability, their gene expression profile was distinct from the established nT reg “genetic signature,” indicative of developmental and possibly mechanistic differences. These results identified a functional role for iT reg cells in vivo and demonstrated that both iT reg and nT reg cells can act in concert to maintain tolerance.