The aorta-gonad-mesonephros region, from which definitive hematopoiesis first arises in midgestation mouse embryos, has intra-aortic hematopoietic clusters (IAHCs) containing hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We previously reported expression of the transcription factor Sox17 in IAHCs, and overexpression of Sox17 in CD45c-KIT cells comprising IAHCs maintains the formation of cell clusters and their multipotency in vitro over multiple passages. Here, we demonstrate the importance of NOTCH1 in IAHC formation and maintenance of the HSC/HPC phenotype. We further show that Notch1 expression is positively regulated by SOX17 via direct binding to its gene promoter. SOX17 and NOTCH1 were both found to be expressed in vivo in cells of IAHCs by whole mount immunostaining. We found that cells transduced with the active form of NOTCH1 or its downstream target, Hes1, maintained their multipotent colony-forming capacity in semisolid medium. Moreover, cells stimulated by NOTCH1 ligand, Jagged1, or Delta-like protein 1, had the capacity to form multilineage colonies. Conversely, knockdown of Notch1 and Hes1 led to a reduction of their multipotent colony-forming capacity. These results suggest that the Sox17-Notch1-Hes1 pathway is critical for maintaining the undifferentiated state of IAHCs.