Notch signaling is required for multiple aspects of cardiovascular development, including arterial-venous differentiation, septation and cushion formation. Despite recognition of the importance of the Notch pathway in normal cardiovascular development, the proximate downstream effectors are not yet known. Likely candidate effectors are members of the hairy and enhancer of split related (hesr) family of bHLH transcription factors. However, mutational analysis of individual hesr genes has so far failed to elucidate their role in all Notch-mediated cardiovascular signaling events. An example of this is evident for mutants of gridlock, the zebrafish counterpart of mouse hesr2, which have vascular defects, whereas mouse hesr2 mutants have only cardiac defects. One possible explanation for these differences could be functional redundancy between hesr family members. Here, we report that mice lacking the hesr1 gene are viable and fertile, whereas knockout mouse of both hesr1 and hesr2 is embryonic lethal at 11.5 days postcoitum (dpc) and recapitulates most of the known cardiovascular phenotypes of disrupted Notch pathway mutants including defects in arterial-venous specification, septation and cushion formation. Taken together, our results demonstrate a requirement for hesr1 and hesr2 in mediating Notch signaling in the developing cardiac and vascular systems.