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Although there is a criticism that embryonic stem (ES) cell differentiation does not always reflect the differentiation process involved in mouse development, it is a suitable model system to dissect the specific differentiation pathway. We established the culture conditions that selectively differentiated mouse ES cells into three germ layers containing mesendoderm, definitive endoderm (DE), visceral endoderm (VE), mesoderm, and neuroectoderm. However, the molecular mechanisms of differentiation under each specific condition still remain unclear. Here, in combination with the RNA interference‐mediated gene knockdown (KD) method, we show that  Eomesodermin  ( Eomes ),  Mixl1 ,  Brachyury  ( T ), and  GATA6  are major molecular determinants in the differentiation of mesendoderm, DE, VE, and mesoderm.  Eomes  plays a pivotal role in an early stage of mesendoderm differentiation, whereas  Mixl1  does the same in the later stage where mesendoderm differentiates into DE. Further analyses of quantitative reverse transcription polymerase chain reaction and overexpression of  Mixl1  demonstrated that  Mixl1  is genetically a downstream molecule of  Eomes . In addition, both  Eomes  and  Mixl1  act as negative regulators of  T  expression. This strategy also reveals that  Eomes  and  T  play cell‐autonomous roles in platelet‐derived growth factor receptor α (PDGFRα) +  vascular endothelial growth factor receptor 2 (VEGFR2) +  and PDGFRα +  mesoderm generations, respectively. Our results obtained from this study are fully consistent with previous knockout studies of those genes. The present study, therefore, demonstrates that the major molecular mechanism underlying in vitro ES cell differentiation largely recapitulates that in actual embryogenesis, and the combination of our culture system and RNAi‐mediated gene KD is an useful tool to elucidate the molecular hierarchy in in vitro ES cell differentiation.  Disclosure of potential conflicts of interest is found at the end of this article.

Dissecting the Molecular Hierarchy for Mesendoderm Differentiation Through a Combination of Embryonic Stem Cell Culture and RNA Interference