Regulatory T cells play a central role in controlling homeostasis, and in inducing and maintaining tolerance to both foreign and self-antigens. Several types of T cells with regulatory activity have been described both in mice and humans, and those within the CD4+ subset have been extensively studied. Among them, the best characterized are the naturally occurring CD4+ CD25+ regulatory T (Treg) cells, and the adaptive type 1 regulatory T (Tr1) cells. Natural Treg cells can arise directly from the thymus, are characterized by the constitutive expression of the transcription factor Foxp3, and suppress T cell responses in a cell-cell contact mediated mechanism. On the contrary, adaptive Tr1 cells arise in the periphery upon encountering antigen in a tolerogenic environment, produce high levels of interleukin (IL)-10 and mediate suppression via IL-10. During the last decade, much effort has been placed on developing protocols to generate regulatory T-cell lines and clones, to further define the similarities and differences between various regulatory T-cell subsets. In this chapter, we will outline protocols to expand naturally occurring Treg cells, to differentiate homogeneous population of Tr1 cells in vitro, and to generate natural Treg and Tr1 cell clones and cell lines.