Abstract Background Identifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength. Here we examine development in embryos expressing various combinations of Notch1 mutant alleles. Mice homozygous for the hypomorphic Notch1 12 f allele, which removes the single O -fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch1 ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Mice homozygous for the inactive Notch1 lbd allele express Notch1 missing an ~20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day ~E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch1 12 f / lbd embryos were compared with Notch1 +/12 f , Notch1 12 f /12 f , and Notch1 lbd / lbd embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch1 12 f / lbd compound heterozygotes compared to Notch1 lbd / lbd embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch1 12 f and a null Notch1 allele. Results Mouse embryos expressing the hypomorphic Notch1 12 f allele, in combination with the inactive Notch1 lbd allele which lacks the Notch1 ligand binding domain, died at ~E11.5-12.5. Notch1 12 f / lbd ES cells signaled less well than Notch1 12 f /12 f ES cells but more strongly than Notch1 lbd / lbd ES cells. However, vascular defects in Notch1 12 f / lbd yolk sac were severe and similar to Notch1 lbd / lbd yolk sac. By contrast, vascular disorganization was milder in Notch1 12 f / lbd compared to Notch1 lbd / lbd embryos. The expression of Notch1 target genes was low in Notch1 12 f / lbd yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch1 12 f / lbd yolk sac phenotype suggested that the allelic products may functionally interact. By contrast, compound heterozygotes with Notch1 12 f in combination with a Notch1 null allele ( Notch1 tm1Con ) were capable of surviving to birth. Conclusions Notch1 signaling in Notch1 12 f / lbd compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch1 12 f allele and a Notch1 null allele. The data suggest that the gene products Notch1 lbd and Notch1 12f interact to reduce the activity of Notch1 12f .