Cardiogenesis can be induced in vitro in ES cells, though it is difficult to distinguish cardiac-specific genes, since embryoid bodies simultaneously differentiate into multiple lineages. In the present study, transient serum removal during culture greatly enhanced cardiogenesis, and reduced generation of endothelial and hematopoietic cells. Using DNA microarray analysis of 24 differentiated sample cultures including cardiogenesis-enhanced cells, we successfully selected genes up-regulated in embryoid bodies that had undergone cardiogenic differentiation. Besides contractile protein genes, cardiac transcriptional regulatory genes, such as Nkx2-5, Gata4/5, Mef2c, and Myocd, were primary constituents of the first 100 genes chosen as cardiogenesis-associated genes. Further, whole mount in situ hybridization analysis of 13 genes containing non-characterized ones confirmed that most of them were specifically expressed in the heart region of mouse embryos from E9.5–10.5. Based on our results, we consider that the present profiling method may be useful to identify novel genes important for cardiac development.