To recapitulate this in vitro and generate myelin organoids, the EBs are cultured in media with specific growth factors and supplements that lead to ectodermal differentiation specifically, followed by subsequent neural induction. A well established method used to efficiently differentiate hPSC into neural cells is by dual inhibition of SMAD signaling using dorsomorphin (also known as compound C) and SB431542. To promote further proliferation of neural precursor cells specific growth factors are added to the media such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). Before neural and glial induction, the EBs are generally embedded in an extracellular matrix, such as Matrigel, and transferred to a rotating bioreactor where different small molecules and growth factors are continuously supplemented to promote the differentiation of cells into specific structures and cell types. In vivo, neuronal induction precedes oligodendrocyte formation. Therefore, in culture, neuronal induction factors are added first to form neuroepithelial structures followed by factors that induce oligodendrocyte precursor cell (OPC) formation and differentiation into oligodendroglia. To promote formation of neurons from neural precursor cells, brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) can be added to the media. Subsequently, factors such as Platelet-derived growth factor AA (PDGF-AA) and Insulin-like growth factor 1 (IGF-1) are added to the media to result in an expansion of the OPC populations present within the organoid by promoting OPC proliferation and survival. Finally, factors that induce OPC differentiation into oligodendrocytes, and ultimately myelinating oligodendrocytes, are added. This includes thyroid hormone (T3), which has been shown to induce oligodendrocyte generation from OPCs in vivo. The organoids are maintained in suspension where they grow and mature until required for analysis. The fundamentals of this workflow are generally used to obtain myelin organoids, however, various protocols that rely on it have introduced multiple modifications for different purposes. Madhavan et al. was the first to establish a reproducible protocol that allowed for generating organoids with robust OPC and oligodendrocytes populations, and therefore myelination, they are referred to as myelin organoids, or myelinoids (Fig 1). Properties and Components The generation of myelin organoids generally relies on neurocortical patterning factors that establish the structural and cellular framework necessary for the induction of oligodendrogenesis later on in the differentiation protocol. Therefore, the properties and components of myelin organoids in the early stages of differentiation are very similar to that observed in cerebral organoids where populations of neural progenitor cells,