© Springer Science+Business Media New York 2016. Regulatory T cells (Tregs) that suppress the activation of immune effector cells limit immunopathology and are fast emerging as therapeutic targets for autoimmune and cancer disease. Tools enabling Treg in vitro -induction, expansion, and characterization and manipulation will help future clinical developments. In this chapter, we describe in detail how to use bacterial superantigens to induce human Tregs effi ciently from peripheral blood mononuclear cells. How to assess human Treg phenotype and suppressive capacity are also described. Technical details, variations, and alternative experimental conditions are provided.