Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a muchneeded human gonad organoid for studying disorders/differences of sex development.

While the Hedgehog signalling pathway is implicated in numerous developmental processes and maladies, variants in the <i>Desert Hedgehog</i> (<i>DHH</i>) ligand underlie a condition characterised by 46,XY gonadal dysgenesis with or without peripheral neuropathy. We discuss here the role and regulation of <i>DHH</i> and its signalling pathway in the developing gonads and examine the current understanding of how disruption to this pathway causes this difference of sex development (DSD) in humans.