AbstractMouse embryonic stem (ES) cells are derived from mouse blastocyst and are able to generate all embryonic tissues in vitro. This propensity of ES cells has acquired considerable attention in recent years due to the promising potential for future cell replacement–based therapies. Therefore, it is of fundamental interest to establish protocols that allow the differentiation of ES cells into specific cell types. In recent years, several such differentiation procedures have been described for mouse and human embryonic stem cells. This unit describes a simple procedure that promotes the neuronal differentiation of mouse embryonic stem cells and yields a high proportion of midbrain dopaminergic neurons. Furthermore, this procedure permits the isolation of neural stem cell lines from mouse ES cells. Curr. Protoc. Neurosci. 47:3.6.1‐3.6.21. © 2009 by John Wiley & Sons, Inc.