Introduction: Embryonic stem (ES) cells are pluripotent cells derived from inner cell mass of blastocyst. These cells can product any cell by spontaneous or directed differentiation. This study was started to produce neuron cells by directed differentiation of new murine ES cell line in vitro. Materials and Methods: Murine ES cells (Royan B1 derived from C57BL/6 Strain) were used for in vitro neuron differentiation. After production of embryoid bodies from ES cells, a highly enriched population of neuroepithelial precursor cells were derived from ES cell proliferation in the presence of growth factors (EGF and bFGF). These cells differentiate into both neurons and glia following withdrawal of growth factors and replacement of media. Immunohistochemistry and electrophysiology were used for evaluation of differentiated neurons. Results: Antibodies raised against microtubule associated proteins (MAP2), glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) antigens revealed neuron cells with GABAergic and dopaminergic characteristics. Also, the neurons produced action potentials and responsed to electrical stimulations. Conclusion: Embryonic stem cell of Royan B1 can produce functional neuron cells in vitro, by directed differentiation.