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Summary: Generation of human motor neurons (MNs) overcomes the inaccessibility to patient brain tissues and greatly facilitates the research in MN-related diseases. Here, we describe a protocol for generation of neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs), followed by preparation of functional MNs. The optimized induction condition with the expression of three transcription factors in a single lentiviral vector significantly improved the yield and purity, making it possible to biochemically identify dysregulated factors in diseased neurons.For complete details on the use and execution of this protocol, please refer to Ding (2021), Ding et al. (2021), and Sepehrimanesh and Ding (2020).