Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4, and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerise on adjacent cis regulatory elements, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In this study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy (FCS). We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Sox/Oct motif of Fgf4. Taking into account that Sox2 expression levels fluctuate more than Oct4, our finding could explain how Sox2 controls the segregation of the cells of the inner cell mass (ICM) into the epiblast (EPI) and the primitive endoderm (PE) populations within the developing rodent blastocyst.