NKT cell subsets can be divided based on CD4 and NK1.1 expression and tissue of origin, but the developmental and functional relationships between the different subsets still are poorly understood. A comprehensive study of 19 cytokines across different NKT cell subsets revealed that no two NKT subpopulations exhibited the same cytokine profile, and, remarkably, the amounts of each cytokine produced varied by up to 100-fold or more among subsets. This study also revealed the existence of a population of CD4⁻NK1.1⁻ NKT cells that produce high levels of the proinflammatory cytokine IL-17 within 2-3 h of activation. On intrathymic transfer these cells develop into mature CD4⁻NK1.1⁺ but not into CD4⁺NK1.1⁺ NKT cells, indicating that CD4⁻NK1.1⁻ NKT cells include an IL-17-producing subpopulation, and also mark the elusive branch point for CD4⁺ and CD4⁻ NKT cell sublineages.