The pluripotent state is traditionally associated with large absolute levels of certain transcription factors such as Nanog and Oct4. Here we present experimental observations using quantitative immunofluorescence, that pluripotency in mouse embryonic stem (mES) cells is established by specific ratios between Oct4 and Nanog. When cells are grown in 2i conditions they exhibit uniform levels of pluripotency and this is associated with a high correlation between the levels of Oct4 and Nanog in individual cells. The correlation is lost when cells differentiate. Our results suggest that the correlation between these two factors and the distribution of Oct4:Nanog ratios can be used as quantifiers to distinguish between three sub-populations in an mES cell culture: pluripotent, lineage-primed and differentiating cells. When we apply these quantifiers to cells with lower levels of Nanog or mutant for β-catenin or Tcf3, the results suggest that these cells exhibit higher probability of differentiation.