Epicardium-derived cells (EPDCs) contribute to formation of coronary vessels and fibrous matrix of the mature heart. Nuclear factor of activated T-cells cytoplasmic 1 (NFATC1) is expressed in cells of the proepicardium (PE), epicardium and EPDCs in mouse and chick embryos. Conditional loss of NFATC1 expression in EPDCs in mice causes embryonic death by E18.5 with reduced coronary vessel and fibrous matrix penetration into myocardium. In osteoclasts, calcineurin-mediated activation of NFATC1 by receptor activator of NFκB ligand (RANKL) signaling induces cathepsin K (CTSK) expression for extracellular matrix degradation and cell invasion. RANKL/NFATC1 pathway components also are expressed in EPDCs, and loss of NFATC1 in EPDCs causes loss of CTSK expression in the myocardial interstitium in vivo. Likewise, RANKL treatment induces Ctsk expression in PE-derived cell cultures via a calcineurin-dependent mechanism. In chicken embryo hearts, RANKL treatment increases the distance of EPDC invasion into myocardium, and this response is calcineurin dependent. Together, these data demonstrate a crucial role for the RANKL/NFATC1 signaling pathway in promoting invasion of EPDCs into the myocardium by induction of extracellular matrix-degrading enzyme gene expression.