Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1⁺/Oct3/4⁺ cells and Rex1^-/Oct3/4⁺ cells. The Rex1^-/Oct3/4⁺ and Rex1⁺/Oct3/4⁺ populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1⁺/Oct3/4⁺ cells and Rex1^-/Oct3/4⁺ cells have characteristics similar to those of ICM and early-PrE cells, Rex1⁺/Oct3/4⁺ cells predominantly differentiated into primitive ectoderm and contributed to chimera formation,whereas Rex1^-/Oct3/4⁺ cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.