Endocardial cells and cardiomyocytes differentiate from the cardiogenic mesoderm at about the same time during development. Although in vitro embryonic stem (ES) cell systems have been used to study the differentiation of various types of cell lineages, including cardiomyocytes, smooth muscle cells, and vascular endothelial cells, differentiation of endocardial cells, or endocardiogenesis, has not been well reported, because of a lack of specific molecular markers. In our search for cardiogenesis-associated genes expressed in embryoid bodies, we found several genes expressed in the heart region of mouse embryos, but not in cardiomyocytes. To identify the cell types expressing these genes, CD31(+) cells were taken from mouse embryos on embryonic day (E)8.5 and E9.5 and sorted, then their transcripts were analyzed using quantitative RT-PCR analyses. In those embryos, Gata4 and Nfatc1, as well as newly identified Cgnl1 and Dok4 were found to be preferentially expressed in endocardial cells, but not in yolk sac endothelial cells, while Cdh5 and Kdr were expressed in both cardiac and yolk sac endothelial cells. Immunohistochemical analyses of embryoid bodies revealed that some CD31(+) cells co-expressing Gata4 and Nfatc1 were located in close proximity to cardiomyocytes. These results suggest that embryoid bodies express endocardial specific genes and likely generate endocardial cells along with cardiomyocytes. Further, they indicate that these new marker genes are useful to study the origin and induction of endocardial cells, and identify other endocardial markers.