Techniques for controlling the expression of a specific gene in embryonic stem cells are effective and important for clarifying the functions of the gene. Regarding differentiation of cells into nervous system components, these techniques would play key roles in elucidating, not only the differentiation mechanisms of neuronal and glial cells, but also how neuronal phenotypes are determined. In this chapter, we describe an RNA interference method for suppressing cholinergic differentiation in murine embryonic stem cells by knockdown of expression of the transcription factor L3/Lhx8, a Lim homeobox gene family protein. This method will greatly facilitate functional analyses of the factors involved in neuronal differentiation and regeneration and contribute to cell transplantation studies.
