Vitamin A (retinol [Rol]) and its metabolites are essential for embryonic development. The Rol metabolite all-trans retinoic acid (RA) is a biologically active form of Rol. The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription-factors (COUP-TF) proteins have been implicated in the regulation of several important biological processes, such as embryonic development and neuronal cell differentiation. Because there is evidence that COUP-TFs function in the retinoid signaling network during development and differentiation, we generated murine embryonic stem (ES) cell lines which stably and constitutively overexpress COUP-TF1 (NR2F1) and we analyzed RA-induced differentiation. COUP-TF1 overexpression resulted in reduced RA-associated growth arrest. A 2.4+/-0.17-fold higher Nanog mRNA level was seen in COUP-TF1 overexpressing lines, as compared with wild-type (WT) ES cells, after a 72 hr RA treatment. We also showed that COUP-TF1 overexpression enhanced RA-induced extraembryonic endoderm gene expression. Specifically, COUP-TF1 overexpression increased mRNA levels of GATA6 by 3.3+/-0.3-fold, GATA4 by 3.6+/-0.1-fold, laminin B1 (LAMB1) by 3.4+/-0.1-fold, LAMC1 by 3.4+/-0.2-fold, Dab2 by 2.4+0.1-fold, and SOX17 by 2.5-fold at 72 hr after RA treatment plus LIF, as compared with the increases seen in WT ES cells. However, RA-induced neurogenesis was unaffected by COUP-TF1 overexpression, as shown by the equivalent levels of expression of NeuroD1, nestin, GAP43 and other neuronal markers. Our results revealed for the first time that COUP-TF1 is an important signaling molecule during vitamin A (Rol)-mediated very early stage of embryonic development.