NFATc1 transcription factor is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. We identified a role for calcineurin-NFAT signaling in lymphatic development and patterning. NFATc1 was colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin on cardinal vein as lymphatic endothelial cells (LEC) are specified and as they segregate into lymph sacs and mature lymphatics. In NFATc1 null mice, Prox-1, VEGFR-3 and podoplanin positive endothelial cells sprouted from the cardinal vein at E11.5, but poorly coalesced into lymph sacs. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with the calcineurin inhibitor cyclosporine-A showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by the lymphatics and irregular patterning of the LEC sprouts coming off the jugular lymph sac, which suggests a role for calcineurin-NFAT signaling in lymphatic patterning. In a murine model of injury-induced lymphangiogenesis, NFATc1 was expressed on the neolymphatics induced by lung-specific overexpression of VEGF-A. Mice lacking the calcineurin Abeta regulatory subunit, with diminished nuclear NFAT, failed to respond to VEGF-A with increased lymphangiogenesis. In vitro, endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression by human microvascular endothelial cells was reduced by siRNA to NFATc1, to levels comparable to reductions seen with siRNA to Prox-1. In reporter assays, NFATc1 activated lymphatic specific gene promoters. These results demonstrate the role of calcineurin-NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved.