Invariant natural killer T (iNKT) cells constitute a subpopulation of T cells that recognize glycolipids presented by CD1d molecules. They are characterized by their prompt production of interleukin-4 (IL-4) and interferon- (IFN- ), which enables them to modulate diverse immune responses. Recently, we enlarged this concept by identifying a distinct IL-17-producing iNKT cell subset, named iNKT17 cells. The mechanisms leading to the acquisition of this new iNKT cell activity are unknown. Herein we show that IL-17-producing iNKT cells are already present in the thymus, predominantly among a subset regarded so far as an immature stage of thymic iNKT cell development, the CD1d tetramerposCD44posNK1.1negCD4neg cells. Using EGFP reporter mice, we demonstrate that the transcription factor ROR- t is critical for the thymic differentiation of this subset because only ROR- tpos iNKT cells are capable of massively secreting IL-17. Moreover, IL-17-producing CD1d tetramerposCD44posNK1.1negCD4neg thymic iNKT cells have reached a mature differentiation stage because they fail to generate other cell subsets in fetal thymic organ culture. Conversely, thymic ROR- tneg iNKT cell precursors give rise to progeny, but acquire neither ROR- t expression nor the ability to secrete IL-17. In conclusion, our findings demonstrate an alternative thymic pathway leading to the development of iNKT17 cells that requires ROR- t expression.