The therapeutic potential of embryonic stem (ES) cells is promising, but in many cases limited by our inability to promote their differentiation to specific cell types, such as motor neurons. Here we provide the first report of the successful differentiation of human ES cells to cells of a motor neuron phenotype. A renewable source of neuroepithelial cells was generated from human ES cells. Extracellular signals were then employed to induce motor neuron differentiation and related gene expression by these cells. OLIG2 and HLXB9 gene expression increased upon the addition of basic fibroblast growth factor, retinoic acid, and sonic hedgehog, as a motor neuron phenotype expressing Islet1 and choline acetyltransferase (ChAT) developed. This study demonstrates that neuroepithelial cells derived from human ES cells are renewable progenitors capable of generating motor neurons at levels that may be therapeutically useful. Sonic hedgehog, basic fibroblast growth factor, and retinoic acid differentially influence human motor neuron differentiation by mechanisms that remain to be defined.