Myelination is essential for central nervous system (CNS) formation, health, and function. Emerging evidence of oligodendrocyte heterogeneity in health and disease and divergent CNS gene expression profiles between mice and humans supports the development of experimentally tractable human myelination systems. Here, we developed human iPSC-derived myelinating organoids (“myelinoids”) and quantitative tools to study myelination from oligodendrogenesis through to compact myelin formation and myelinated axon organization. Using patient-derived cells, we modeled a monogenetic disease of myelinated axons (Nfasc155 deficiency), recapitulating impaired paranodal axo-glial junction formation. We also validated the use of myelinoids for pharmacological assessment of myelination-both at the level of individual oligodendrocytes and globally across whole myelinoids-and demonstrated reduced myelination in response to suppressed synaptic vesicle release. Our study provides a platform to investigate human myelin development, disease, and adaptive myelination.