To evaluate interactions between human endothelial cells (ECs) and smooth muscle cells (SMCs) for the development of tissue-engineered vessels, we examined the adhesion and key cell properties of human ECs grown on quiescent human aortic SMCs. ECs attached to SMCs spread more slowly than ECs attached to fibronectin surfaces, and ECs aligned along the direction of the SMCs. ECs attached firmly and less than 5% of the cells were removed by shear stresses as high as 300 dyn cm⁻². Unlike porcine SMCs and co-cultures, human SMCs or co-cultures do not contract under flow, and the human ECs and SMCs in co-culture align toward the direction of flow. A confluent endothelium could be maintained in co-culture for over 30 days, and some of the ECs reoriented perpendicular to the SMCs after 9 days in static culture. Surface tissue factor levels in ECs and SMCs were less in co-culture than in monoculture. Co-culture induced an increase in calponin expression in SMCs. These findings show that human co-cultures can be maintained for long culture periods, where the endothelium remains confluent and responds to long-term exposure to flow, and EC–SMC interactions lead to an increase in SMC differentiation and an EC surface that is less thrombotic.