CD4+CD25+ regulatory T cells (Tregs) are crucial for the maintenance of immunological tolerance. Recent data indicate that Tregs not only develop in the thymus during ontogeny but can also differentiate from naive T cells in the periphery. The following protocol describes a method by which Tregs are generated in vitro by stimulation of naive T cells in the presence of transforming growth factor β (Ti-Tregs). In vitro-induced regulatory T cells express markers of conventional Treg such as CD25 and the genetic program committing transcription factor FoxP3. Functionally the in vitro-generated Ti-Tregs suppress T-cell activation and proliferation while in vivo these cells have been proven to control inflammation in different animal models, suggesting a potential use of these cells for immunotherapy. The protocol can be completed within 5 days.