The natriuretic peptide family consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). We have elucidated that CNP is synthesized by endothelial cells. We have also shown that CNP secretion is potently suppressed by vascular endothelial growth factor (VEGF). In the present study, we examined the developmental gene expression of the natriuretic peptide system with the expression of VEGF and endothelial cell-specific receptor tyrosine kinases (RTKs), which expression is necessary for vasculogenesis, using embryoid bodies (EB) as an in vitro model for vascular development. When mouse embryonic stem (ES) cells were cultured in suspension culture, ES cells spontaneously differentiated into EB on day 4 and then into cystic EB (day 10). The VEGF gene transcript was detected early, on day 4. The expression of Flk-1, and flt-1 (the two VEGF receptors) and also of tie-2, which is crucial for blood island formation, was detected as early as day 4, and also on days 8 and 21. In contrast, the expression of flt-4, the receptor for VEGF-C, and tie-1, was first detected on day 21. Along with the developmental expression of these markers of differentiation for endothelial cells, the gene expression of CNP and its specific receptor, ANP-B receptor, was detected on days 4, 8, and 21. In contrast, the gene expression of BNP, which acts as a cardiac hormone, and the gene expression of the ANP-A receptor, which is specific to ANP and BNP, was first detected on days 8 and 21, respectively. These results indicate the distinct role of CNP in the natriuretic peptide family and the close linkage of CNP expression and endothelial cell differentiation, suggesting a possible role of CNP in vasculogenesis.