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In the CNS, oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust, fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined, growth factor–rich medium. Within 8 d, PSCs differentiate into paired box 6–positive (PAX6⁺) neural stem cells, which give rise to OLIG2⁺ progenitors by day 12. Oligodendrocyte lineage transcription factor 2–positive (OLIG2⁺) cells begin to express the transcription factor NKX2.2 around day 18, followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4⁺) appear around day 50 and reach, on average, 43% of the cell population after 75 d of differentiation. O4⁺ OPCs can be …