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Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse; however, it remains unknown how  cyclin D1  expression is regulated during early embryonic development. Here, we define the role of germ cell nuclear factor ( GCNF ) in the activation of cyclin D1 expression during embryonic stem cell (ESC) differentiation as a model of early development. During our study of  GCNF  knockout ( GCNF −  /  −  ) ESC, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ESC differentiation. This was determined to be an indirect effect of deregulation Mir302a, which is a cyclin D1 suppressor via binding to the 3′UTR of cyclin D1 mRNA. Moreover, we showed that  Mir302  is a target gene of GCNF that inhibits  Mir302  expression by binding to a DR0 element within its promoter. Inhibition of  Mir302a  using Mir302 inhibitor during differentiation of  GCNF −  /  −   ESCs restored cyclin D1 expression. Similarly over‐expression of GCNF during differentiation of  GCNF −  /  −   ESCs rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a. S tem  C ells   2014;32:1527–1537

GCNF‐Dependent Activation of Cyclin D1 Expression via Repression of Mir302a During ESC Differentiation