Background: The forkhead transcription factor FoxO1 is involved in cell cycle regulation during cardiovascular development. Systemic loss of FoxO1 results in lethality at embryonic day 10.5 with severe cardiovascular defects; however, the cell-type-specific requirements for FoxO1 in cardiovascular development are unknown. Here we examine the role of FoxO1 using a conditional loss of function approach. Results: Loss of FoxO1 in differentiated cardiac myocytes has no apparent effect on cardiovascular development. In contrast, endothelial-specific FoxO1 deficiency in Tie2Cre;FoxO1fl/fl embryos results in lethality at E10.5, which recapitulates the FoxO1-null phenotype. Tie2Cre;FoxO1fl/fl embryos have an intact differentiated endothelium, but display defective remodeling of vasculature. Additional effects on heart development include reduced myocardial trabeculation, which is likely secondary to the endothelial abnormalities, and hypoplasia of endocardial cushions. Conclusions: The phenotype of Tie2Cre;FoxO1fl/fl mutant embryos demonstrates that FoxO1 is required specifically in endothelial cells to regulate formation of the heart and vasculature during development. Developmental Dynamics 241:803–813, 2012. © 2012 Wiley Periodicals, Inc.