Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proclaimed as a source of undifferentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson’s disease, Fanconi’s anemia and diabetes. In addition to their potential in regenerative therapy, an understanding of the mechanisms by which these cells differentiate into any functional cell type will provide valuable information about basic biology. Screens for small compounds that can drive self-renewal maintenance or differentiation protocols are relevant to this goal. Nitric oxide (NO) is a diffusible second messenger implicated in numerous physiological functions in mammals. This molecule plays an important role in the maintenance of key features required for embryonic development and extension in ES cells. The goal of this chapter is to discuss recent advances concerning the ways in which NO signaling pathways mediate diverse mechanisms involved in the differentiation of ES cells toward multiple lineages. This chapter will also discuss the mechanisms by which NO can modify tissue-specific gene expression thorough chromatin remodeling and post-translational modification of transcription factors.