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T‐box 3 (Tbx3) is a member of the T‐box family of genes. Mutations that result in the haploinsufficiency of  TBX3  cause ulnar mammary syndrome in humans characterized by mammary gland hypoplasia as well as other congenital defects. In mice, homozygous mutations are embryonic lethal, suggesting that Tbx3 is essential for embryo development. Studies in mice have shown that Tbx3 is essential in the maintenance of mouse embryonic stem cell (ESC) self‐renewal and in their differentiation into extraembryonic endoderm (ExEn). The role TBX3 plays in regulating human ESCs (hESCs) has not been explored. Since mouse and hESCs are known to represent distinct pluripotent states, it is important to address the role of TBX3 in hESC self‐renewal and differentiation. Using overexpression and knockdown strategies, we found that TBX3 overexpression promotes hESC proliferation possibly by repressing the expression of both  NFκBIB  and  p14   ARF  , known cell cycle regulators. During differentiation,  TBX3  knockdown resulted in decreased neural rosette formation and in decreased expression of neuroepithelial and neuroectoderm markers ( PAX6 ,  LHX2 ,  FOXG1 , and  RAX ). Taken together, our data suggest a role for TBX3 in hESC proliferation and reveal an unrecognized novel role of TBX3 in promoting neuroepithelial differentiation. Our results suggest that TBX3 plays distinct roles in regulating self‐renewal and differentiation in both hESCs and mouse ESCs. S TEM  C ells  2012;30:2152–2163

TBX3 Promotes Human Embryonic Stem Cell Proliferation and Neuroepithelial Differentiation in a Differentiation Stage‐dependent Manner