T cell fate and function are determined by intricate coordination between cellular signaling and metabolic pathways, but the precise metabolic determinants for iNKT cell development are unknown. We found that iNKT cell development requires intact mitochondrial metabolism. Mice with a T cell-specific deletion of Ubiquinol-Cytochrome C Reductase Rieske Iron-Sulphur Polypeptide 1 (T-Uqcrfs1^-/-), a mitochondrial complex III protein, had a cell-intrinsic defect in iNKT cell development while thymic development of conventional T cells proceeded normally.  Residual iNKT cells in T-Uqcrfs1^-/- mice retained the ability to proliferate in vivo but exhibited increased apoptosis, diminished T-bet levels and impaired responsiveness to TCR and IL-15 stimulation. In the iNKT cell hybridoma cell line DN32.D3 with knockdown of Uqcrfs1, the production of IL-2 was decreased; this was associated with a reduced level and impaired nuclear translocation of NFAT. Interestingly, the development of MAIT cells is also impaired in T-Uqcrfs1^-/- mice. Our study highlights the critical role played by mitochondrial metabolism in providing signaling intermediates that modulate TCR signaling in vivo and their effects on iNKT and MAIT cell development and function.