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Human embryonic stem cell (ESC) pluripotency is thought to be regulated by several key transcription factors including OCT4, NANOG, and SOX2. Although the functions of OCT4 and NANOG in human ESCs are well defined, that of SOX2 has not been fully characterized. To investigate the role of SOX2, we modulated the level of SOX2 expression in human ESCs. Reduction of SOX2 expression in human ESCs induced trophectodermal and partial endodermal differentiation. Interestingly, CDX2, a typical trophectoderm-associated gene, was not up-regulated. In contrast, using the Tet-on gene inducible system, SOX2 over-expression in human ESCs induced trophectoderm differentiation accompanied by increased CDX2 expression. Additionally, SOX2 over-expression resulted in an increase in CGα-positive cells, which marks later stage trophectoderm development, rather than placental lactogen-positive cells. Thus, over-expression as well as repression of SOX2 expression in human ESCs resulted in their differentiation into the trophectoderm lineage. Our data show that SOX2 plays an important role in the maintenance of pluripotency of human ESCs and possibly, trophoblast development.