The applicability of human embryonic stem cells (hESCs) will be greatly enhanced by techniques that permit efficient genetic modification with multiple transgenes. We report here on single-promoter-driven foot-and-mouth disease virus segment 2A-mediated multicistronic expression of a transgene in hESCs. Efficient multicistronic expression of the transgene was permitted by 2A-mediated separation with almost the same amounts of encoded proteins in hESC. In addition, the multicistronic protein expression was successful in hESC-derived differentiated cells in in vivo and in vitro differentiation assays. This technology may be a significant advance in the genetic engineering of hESCs and hESC-derived cells for purposes that require the reliable expression of multiple transgenes. Disclosure of potential conflicts of interest is found at the end of this article.