One of the greatest challenges in regenerative medicine is providing a significant source of vascularization within engineered tissues. Successful vascularization requires both a scaffold that supports vessel formation and a reliable source of vascular cell types. Broad potential for differentiation, high proliferation rates, and autologous availability for neonatal applications make amniotic fluid-derived stem cells (AFSC) well suited for regenerative medicine strategies. We utilized chemical-mediated differentiation of AFSC into endothelial-like cells (AFSC-EC), which expressed key proteins and functional phenotypes associated with endothelial cells. Fibrin-based hydrogels were shown to stimulate AFSC-derived network formation in vitro but were limited by rapid degradation. Incorporation of poly(ethylene glycol) (PEG) provided mechanical stability while retaining key benefits of fibrin-based scaffolds – quick polymerization, high biocompatibility, and vasculogenic stimulation. AFSC-EC as a vascular cell source and AFSC as a perivascular cell source were compared to established sources of these cell types – human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), respectively. In vitro, cell-seeded hydrogels were assessed based on network formation, including parameters such as vessel thickness, length, and area. The development of robust vessels required the presence of both an endothelial and a perivascular cell source and was seen in AFSC co-cultures. Additionally, the co-culture of AFSC with AFSC-EC resulted in a synergistic effect on network parameters similar to MSC. Based on this data, we hypothesized that subcutaneously injecting similar hydrogels in immunodeficient mice would both induce a fibrin-driven angiogenic host response and promote in situ AFSC-derived neovascularization. Two weeks post-injection, AFSC-seeded hydrogels demonstrated significantly higher vascular lumen formation versus those without cells or those seeded with endothelial cells alone; a subset of these lumen were characterized by the presence of red blood cells, suggesting anastamosis with host vasculature. In support of the Pediatric Cardiovascular Bioengineering Lab’s global vision, this research demonstrates that AFSC-seeded fibrin/PEG hydroge In support of the Pediatric Cardiovascular Bioengineering Lab’s global vision, this research demonstrates that AFSC-seeded fibrin/PEG hydrogels have the potential to serve as a vascularized platform for the development of an engineered cardiac patch to be used in autologous repair of congenital heart defects.