Human embryonic stem (hES) cells may generate all major somatic cell types, yet no neuronal subtype has yet been specifically generated in useful purity from hES culture. We report here the selective induction and isolation of functional spinal motor neurons (MNs) from human ES cells. hES cells of the H1 line were transfected with plasmids encoding GFP placed under the control of an MN-specifying enhancer within the 5′-regulatory region of the gene encoding the transcription factor Hb9 and treated with sonic hedgehog (Shh) and retinoic acid (RA). As MNs were induced under the influence of Shh and RA, they activated Hb9-driven GFP expression, permitting their isolation by fluorescence-activated cell sorting (FACS). The MNs thereby generated and isolated became cholinergic and achieved functional maturation in vitro, as evidenced by their fast sodium currents and action potentials on whole-cell patch-clamp and α-bungarotoxin-identified clustering of AChR receptors on co-cultured skeletal myoblasts. The serial combination of these two approaches, motor neuron phenotypic induction followed by Hb9 enhancer-based FACS, permitted the high-efficiency induction and isolation of functional motor neurons from hES cells. These results suggest the utility of promoter/enhancer-based FACS for the isolation of specific phenotypes from hES cell populations as a means of purifying clinically appropriate vectors for cell therapy.