ABSTRACT/PROJECT SUMMARY The adrenal glands (AG) are critical endocrine organs that control the body’s response to stressors, metabolic challenges, blood pressure changes and immune system regulation. AG disease can be caused by many ailments, including genetic faults, malignancies and toxins, and both hyper- and hypofunction of the AGs can be lethal. The AG consists of a core called the medulla that is surrounded by a three-layered cortex, all wrapped in a capsule. It is known that the AGs develop from progenitors from two independent germ layers, i.e. ectoderm/neural crest cells giving rise to the AG medulla and mesoderm/intermediate mesoderm giving rise to the AG cortex. The AG undergoes dynamic cellular homeostasis throughout life driven by adult stem cells located in the capsule. Several open questions remain about the events leading to proper embryonic development of the human AGs, about their transformation and dysfunction in human disorders and about normal adult human homeostasis. Additionally, healthy human AG tissue might become crucial for transplantation approaches for the future treatment of AG disorders. The lack of a human experimental model system that can mimic the complexity of the human organ is causing a slow progression in answering such questions. 3D organoids, derived from human pluripotent stem cells (hPSCs) are ideal to address this gap, however no AG organoids have been reported to date. Here, we propose the generation of complex, assembled AG organoids from both neural crest and intermediate mesoderm progenitors. We will characterize the AG organoids in vitro and upon xenotransplantation based on molecular and functional entities. We will employ the AG organoids to ask questions about human embryonic development and assess their character on the single cell level. Our AG organoids will provide a platform to investigate future questions about AG biology and development, AG disease mechanisms, and ultimately provide the cellular material for cell therapy approaches for AG insufficiency.