This protocol describes methods to identify, purify and culture CD1d restricted invariant natural killer T (iNKT) cells from mouse tissue or human blood samples. The methods for identification and purification of iNKT cells are based on the interaction between iNKT cell receptor and its ligand. The iNKT cell receptor is composed of the invariant Vα14Jα18/Vβ8.2 in mice or Vα24Jα18/Vβ11 in humans and is expressed only on iNKT cells but not on conventional T cells. The iNKT cell antigen receptor in both species recognizes α-galactosylceramide (α-GalCer) presented by the MHC class I-like CD1d. Thus, α-GalCer-loaded CD1d dimer can be used for analysis and purification by fluorescence-activated cell sorting (FACS). Isolation of 1 × 106 purified iNKT cells from mouse thymus, spleen or liver requires 5–6 mice and takes 1–2 h for mononuclear cell preparation from mouse tissues, 1.5 h for enrichment by magnetic beads and 4 h for detection and purification of the iNKT cells by FACS. In the case of isolation of human peripheral blood mononuclear cells (PBMCs) from whole blood, it takes 2 h and requires 5 ml of blood to obtain 5 × 106 PBMCs, which contain 500–25,000 iNKT cells.