NKT cells are innate-like αβ T cells that are conserved between humans and mice. They are distinct from conventional T cells as they recognize lipid antigens presented by the CD1d molecule. Most NKT cells expressed a highly restricted TCR repertoire and can be activated by α-galactosylceramide (α-GalCer) and detected by α-GalCer-loaded-CD1d tetramers. Upon activation, NKT cells respond in few hours by producing cytokines and stimulating many other cells of the innate and adaptive immune system. Over the last decade, many studies have analyzed the regulatory role of NKT cells that can either suppress or exacerbate immune functions. This chapter describes the tools and techniques required to study in vivo and in vitro the regulatory role of NKT cells in mouse as well as from human blood.