The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.