This technology includes a system that allows for robust differentiation of human-induced pluripotent stem cells (iPSC) into motor neurons within a time frame of 7 to 10 days. To differentiate the iPSC, a stable transgene is inserted into the CLYBL safe harbor locus in the human genome using TALENs. The transgene allows for doxycycline-inducible expression of the transcription factors (NGN2, ISL1, and LHX3) that are needed for the cells to differentiate to motor neurons. The technology is described in detail in the protocol paper published by Fernandopulle et al, cited below. We knocked in a doxycycline-inducible transgene cassette into the CLYBI safe harbor locus of iPSCs using TALENs. The cassette drives expression of human NGN2, Isl1, and LXZH3, separated by 2A linker peptides. The targeting vector was a heavily-modified version of a CLYBL targeting vector from Mahendra Rao’s lab at the NIH (essentially, only the homology arms of the original vector were used). The plasmids introduced into the WTC11 and then clones that harbored the transcription factor cassette were selected. The system allows for robust differentiation of human induced pluripotent stem cells (iPSC) into motor neuron cells with an efficient time frame of 7 to 10 days. The system also allows for large scale preparation of motor neurons with few media changes. To differentiate the iPSC, a stable transgene is inserted into the CLYBL safe harbor locus in the human genome. The transgene allows for doxycycline inducible expression of the transcription factors (NGN2, ISL1, and LHX3) that are needed for the cells to differentiate to motor neuron.