Emerging evidence suggests that Notch signaling not only regulates biological processes during development but also participates in the pathogenesis of various diseases in adults, including tumor angiogenesis, hematopoietic malignancies, and cardiometabolic syndromes. Notch signaling involves several ligands and receptors that have unique and overlapping functions. Therefore, blocking function of a ligand or receptor with a neutralizing antibody is a useful approach to examine the specific role of each Notch component. In addition, administration of Notch signaling blocking antibodies in experimental animals offers important insights into clinical translation of Notch biology. In this chapter, we describe examples of in vitro and in vivo loss-of-function experiments with blockade of Notch ligands, particularly Delta-like ligand 4 (Dll4).