The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region\u2013specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain\u2013specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in 24 months in culture.IntroductionUnderstanding the development of the human nervous system and elucidating the mechanisms that lead to brain disorders represent some of the most challenging ongoing endeavors in neurobiology. One major obstacle is the restricted access to healthy and diseased human brain tissue for functional molecular and cellular studies. As a result, experimental paradigms have been largely confined to animal models or in vitro cell culture systems that do not fully recapitulate the developmental, architectural, and species-specific aspects of the human brain. Thus, there is a great need for humanderived model systems that recapitulate features of human CNS development and allow for the study of these processes in both healthy and diseased conditions.