Scaffold-guided vascular tissue engineering has been investigated as a means to generate functional and transplantable vascular tissue grafts that increase the efficacy of cell-based therapeutic strategies in regenerative medicine. In this study, we employed confocal microscopy and three-dimensional reconstruction to assess the engraftment and growth potential of vascular cells within an alginate scaffold with aligned pores. We fabricated honeycomb alginate scaffolds with aligned pores, whose surface was immobilized with fibronectin and subsequently coated with matrigel. Endothelial cells were seeded into aligned pore scaffolds in the presence and absence of human smooth muscle cells. We showed that endothelial cells seeded into alginate scaffolds attach on the surface of aligned pores in vitro, giving rise to stable co-cultures of vascular cells. Moreover, the three-dimensional alginate depots containing the cells were exposed to laminar flow in order to recapitulate physiological shear stress found in the vasculature in vivo. After the flow exposure, the scaffold remained intact and some cells remained adherent to the scaffold and aligned in the flow direction. These studies demonstrate that alginate scaffolds provide a suitable matrix for establishing durable angiogenic modules that may ultimately enhance organ revascularization.