Abstract Body: Background: Numb Family Proteins (NFPs), specifically two mammalian homologs Numb (denoted by Nb) and Numblike (denoted by Nl), have been evident to regulate essential cardiac functions during development. Previous studies reported NFPs as integral for epicardial cells (EpiCs) epithelial mesenchymal-transition (EMT). However, whether NFPs in epicardium regulate EpiCs differentiation to affect cardiac morphogenesis have not been explored. In this study, we investigated how NFPs in epicardium regulate EpiCs EMT and differentiation to modulate cardiac development and subsequently affect cardiac maintenance in adult stages. Method: Using mCherry:Numb knockin mouse model we observed enrichment of Numb in epicardium. We deleted Nb and Nl in epicardium via Tbx18^Cre/+ lineage and refer to this as EDKO. We harvested hearts at embryonic day 15.5/18.5 and assessed morphological and mechanistic differences between control and EDKOs. Conventional and Speckle tracking Echocardiography were used to examine adult heart structure and functions. Results: Our findings revealed that EDKO embryonic hearts display specific morphology with extended left ventricles. We found a surprising difference between female and male EDKOs in terms of survival to adulthood. Reduced EpiCs EMT and Cardiac Fibroblasts (CFs) migration distances, changes in CFs distribution have been observed in EDKO developing hearts. Reduced expression of smooth muscle cells (SMCs) and pericytes also was noticed in EDKOs. Strikingly, EDKO embryonic hearts show defects in ventricular compaction, ventricular wall patterning and in cell proliferation. Furthermore, when survived to adult stage, EDKO hearts revealed significant structural and functional changes over their aging. While digging into regulatory pathways, we found changes in Fibroblast growth factor receptors (Fgfrs)/p-Erk pathway and in activation of Notch1 in EDKO hearts. Conclusion: NFPs in epicardium regulate EpiCs differentiation into CFs and SMCs, and regulate ventricular wall morphogenesis during development. NFPs are also required to maintain cardiac structure and function at adult stages. Fgfrs/p-Erk pathway and Notch1 could be the possible mechanism behind cardiac morphogenetic regulation by NFPs in epicardium. Future aims: Further studies will identify downstream targets of NFPs’ regulation of EpiCs differentiation and ventricular wall patterning in embryonic hearts, and mechanistic pathways of epicardial NFPs’ role in adult hearts.