Objective: Motoneurons are specialized class of neural cells essential for control of body movement and loss of these cells cause to creation of wide range of neurological disease. Embryonic stem cells (ESCs) possess promising potential for cell-based therapies of motoneuron diseases. Here, we describe a new in vitro protocol of directed differentiation of human embryonic stem cells (hESCs) into motoneuron cells. Materials and Methods: Differentiation of neural cells was induced in DMEM/f12 media supplemented with noggin, RA and bFGF. Rosette structures were observed 12 days after induction of differentiation and then neural tube-like structures were formed at 18th day. Dissociated neural tube-like structures were treated with SHH and RA for additional 6 days in suspension culture. Sequential stages of differentiation into motorneurons were confirmed with various methods such as Immunostaining, Patch clamp recording, qRT-PCR and flowcytometry. Results: Flowcytometry analysis of differentiated cells at neural ectoderm stage showed high expression of Nestin, Sox1, and Pax6. Differentiated neural cells have the Na, Ca currents and excitatory postsynaptic potentials (EPSPs)-like. Immunofluorescence staining of matured neural cells showed the expression of MAP2, Tuj1, HB9, islet1, choline acetyl transferase and GFAP. Analysis of Motorneuron specific markers by quantitative RT-PCR is ongoing. Conclusion: Taking together these findings suggest that our differentiation protocol has the capacity to generate functional motorneurons.