As defects in fetal adrenal (FAd) development can result in life threatening primary adrenal insufficiency (PAI), understanding the cellular and gene regulatory mechanisms governing this process is essential. While much has been learned about FAd development in rodent models, species-specific developmental differences limit our understanding of this process in humans. As mechanistic evaluation in human embryos is untenable, we recently developed the first human induced pluripotent stem cells (iPSCs)-derived FAd organoid system that recapitulates normal functional development and steroidogenesis. Using this system, we will undertake the first perturbative and reverse genetic assessment of human FAd development, allowing us to elucidate the molecular mechanisms of human adrenocortical development, which has broad implications in providing essential insight into mechanisms driving PAI. Development of the human adrenal cortex starts with specification of the adrenal primordium (AP) from the coelomic epithelium (CE), followed by establishment of the definitive zone (DZ) with putative stem cell/progenitor potential, and the fetal zone (FZ) with steroidogenic potential. Our recent single cell RNA-seq analysis of the human FAd cortex supports FZ replenishment by the DZ and the observed expression of Wnt ligands/activators in the peripherally located capsule (Cap) is suggestive of niche function. To mechanistically assess human FAd development and understand genetic defects (e.g., NR5A1, WNT4 mutations) driving PAI, we utilized our FAd organoid system. While the transcription factor NR5A1 impacts early FAd cell fate and promotes steroidogenesis in a gene-dose dependent manner in mice, its role in human FAd appears more complex. Our preliminary studies show that induced NR5A1 null mutant organoids fail to differentiate into AP-like cells (APLCs), exhibit decreased survival, fail to upregulate the receptor for adrenocorticotropic hormone (ACTH), and that the steroidogenic potential of the remaining cells is limited. In wild-type FAd organoids, we find that formation of DZ-like cells (DZLCs) is enhanced by the removal of Wnt inhibitor/addition of Wnt agonist. We also find that WNT4 and RSPO3 are highly expressed in both human Cap in vivo and Cap-like cells (CapLCs) in FAd organoids. Intriguingly, we find that DZLC formation is enhanced by sonic hedgehog (SHH), a trophic factor for murine Cap, suggesting a potential SHH-driven niche function for the Cap that is further supported by the observed upregulation of SHH target genes in human Cap. Finally, we find that both FACS-sorted DZ and DZLCs readily differentiate into FZ-like cells (FZLCs) upon ACTH stimulation, supporting the ability of DZLCs to replenish FZLCs. However, as Wnt-target gene expression is maintained in the subcapsular DZ despite exposure to ACTH, it suggests that Cap-derived Wnt signaling antagonizes ACTH- mediated differentiation of DZLCs. Using our organoid system, we will test the central h...