This protocol describes the rapid and robust differentiation of hiPSCs into lower motor neurons (i3LMNs) via induced expression of the transcription factors NGN2, ISL1, and LHX3 (hNIL) (Mazzoni et al., 2013; Shi et al., 2017). In particular, a donor construct containing these factors under the tetracycline response element (TRE3G) (Gossen & Bujard, 1992), a CAG promoter driving constitutive expression of the reverse tetracycline transactivator (rtTA3G), and an EF-1α promoter driving constitutive expression of selection genes (mCherry for Addgene, cat. no. 105841 and SBP-LNGFR and mApple for Addgene, cat. no. 105842) was stably integrated into the safe-harbor CLYBL locus via TALENs (Cerbini et al., 2015). Motor neuron differentiation efficiency can vary between iPSC lines, and homozygous insertion into both CLYBL alleles may result in improved efficiency. Reduced differentiation efficiency of mCherry positive clones can occur with off-target integration of the donor construct. Additionally, the mCherry reporter is flanked by loxP sites, permitting excision by transient transfection of Cre recombinase if desired.