CD4⁺CD25⁺FOXP3⁺ T regulatory (Treg) cells are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. The possibility to use Treg cells for the treatment of T-cell-mediated diseases has recently gained increasing momentum. However, given the limited amount of circulating FOXP3⁺ Treg cells, efficient methods for their ex vivo expansion are highly desirable. Rapamycin allows for in vitro expansion of murine and human FOXP3⁺ Treg cells, which maintain their regulatory phenotype and suppressive capacity. Here, we describe in detail the powerful methods for enriching human FOXP3⁺ Treg cells starting from unfractionated CD4⁺ T cells or for expanding CD25⁺-enriched Treg cells in the presence of rapamycin.