In this chapter, we introduce a co-culture protocol for human embryonic stem (hES) cell differentiation in which opamine (DA) neurons with midbrain-specific markers are efficiently derived. Human ES cells on a feeder layer of stromal cells are induced to differentiate into neuroepithelial or neural precursor cells with embryonic midbrain precursor properties. The resulting neural precursor cells are then selectively expanded and serially passaged to obtain a large, homogeneous population of these cells. Under the conditions for terminal differentiation, the majority of hES-derived neural precursors differentiate into neuronal cells that are positive for DA neuronal markers such as tyrosine hydroxylase (TH) and function in vitro as presynaptic DA neurons.