Unlike conventional αβT cells, invariant natural killer T (iNKT) cells complete their terminal differentiation to functionaliNKT1/2/17 cells in the thymus. However, underlying molecular programs that guideiNKT subset differentiation remain unclear. Here, we profiled the transcriptomes of over 17,000iNKT cells and the chromatin accessibility states of over 39,000iNKT cells across four thymiciNKT developmental stages using single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to define their developmental trajectories. Our study discovered novel features foriNKT precursors and differentiNKT subsets and indicated thatiNKT2 andiNKT17 lineage commitment may occur as early as stage 0 (ST0) by two distinct programs, whileiNKT1 commitments may occur post ST0. BothiNKT1 andiNKT2 cells exhibit extensive phenotypic and functional heterogeneity, whileiNKT17 cells are relatively homogenous. Furthermore, we identified that a novel transcription factor,Cbfβ, was highly expressed iniNKT progenitor commitment checkpoint, which showed a similar expression trajectory with other known transcription factors foriNKT cells development,Zbtb16andEgr2, and could directiNKT cells fate and drive their effector phenotype differentiation. Conditional deletion ofCbfβblocked earlyiNKT cell development and led to severe impairment ofiNKT1/2/17 cell differentiation. Overall, our findings uncovered distinctiNKT developmental programs as well as their cellular heterogeneity, and identified a novel transcription factorCbfβas a key regulator for earlyiNKT cell commitment.