FoxP3 is a key transcription factor for the development and function of natural CD4⁺ regulatory T cells (Treg cells). Here we show that human FoxP3⁺CD4⁺ T cells were composed of three phenotypically and functionally distinct subpopulations: CD45RA⁺FoxP3^lo resting Treg cells (rTreg cells) and CD45RA⁻FoxP3^hi activated Treg cells (aTreg cells), both of which were suppressive in vitro, and cytokine-secreting CD45RA⁻FoxP3^lo nonsuppressive T cells. The proportion of the three subpopulations differed between cord blood, aged individuals, and patients with immunological diseases. Terminally differentiated aTreg cells rapidly died whereas rTreg cells proliferated and converted into aTreg cells in vitro and in vivo. This was shown by the transfer of rTreg cells into NOD-scid-common γ-chain-deficient mice and by TCR sequence-based T cell clonotype tracing in peripheral blood in a normal individual. Taken together, the dissection of FoxP3⁺ cells into subsets enables one to analyze Treg cell differentiation dynamics and interactions in normal and disease states, and to control immune responses through manipulating particular FoxP3⁺ subpopulations.