Based on results from experimental animal models, the adoptive transfer of CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) is expected to be efficacious in treating autoimmune and inflammatory diseases, as well as in preventing alloresponses after solid organ or stem-cell transplantation. For potential clinical applications, large numbers of Treg cells in maximum purity will be required to avoid the risk of disease exacerbation by contaminating effector T cells. We have recently described methods for the efficient in vitro expansion of human Treg cells and identified CD4(+)CD25(high)CD45RA(+) T cells as the ideal starting population for the generation of homogeneous and stable Treg cell products. Here, we provide detailed instructions for their identification, isolation, expansion, and functional characterization.