Invariant NKT (iNKT) cells, a key subset of unconventional T cells, play important roles in cancer, inflammation, infection, and autoimmunity. Although many studies have examined the iNKT cell development pathway in mice, the low frequency of these cells in human blood and tissues has hampered similar studies in humans. To overcome this issue, we used MACS-enrichment to isolate large numbers of iNKT cells from the human thymus. We defined a thymic development pathway for human iNKT cells, identifying a four-stage pathway that involves the progressive increase of the transcription factor PLZF. PLZF is absolutely critical for the development of functional iNKT cells and other subsets of unconventional T cells such as MAIT cells and gamma delta T cells in mice. Development of iNKT cells in the human thymus involves the expression of transcription factors required for the production of TNF, IFNg and IL-2. Our work identifies important differences in the thymic development of mouse and human iNKT cells, with human iNKT cells displaying a TH1 phenotype. A comparison of the development pathways of human iNKT cells with MAIT cells and Vd2+ gamma delta T cells highlights important differences with how these unconventional T cells develop. Taken together, our findings may lead to new opportunities to better manipulate iNKT cells to treat human diseases such as cancer.