A major roadblock to successful organ bioengineering is the need for a functional vascular network within the engineered tissue. Here, we describe the fabrication of three-dimensional, naturally derived scaffolds with an intact vascular tree. Livers from different species were perfused with detergent to selectively remove the cellular components of the tissue while preserving the extracellular matrix components and the intact vascular network. The decellularized vascular network was able to withstand fluid flow that entered through a central inlet vessel, branched into an extensive capillary bed, and coalesced into a single outlet vessel. The vascular network was used to reseed the scaffolds with human fetal liver and endothelial cells. These cells engrafted in their putative native locations within the decellularized organ and displayed typical endothelial, hepatic, and biliary epithelial markers, thus creating a liver-like tissue in vitro. Conclusion: These results represent a significant advancement in the bioengineering of whole organs. This technology may provide the necessary tools to produce the first fully functional bioengineered livers for organ transplantation and drug discovery. (HEPATOLOGY 2011;53:604-617)