Whereas high shearing flows are known to induce endothelial cell remodeling, we show here that very low interstitial flow rates trigger endothelial cell morphogenesis in 3D cultures. Interstitial flow is a functionally critical component of the circulation, and we have recently observed that it plays a regulatory role in lymphangiogenesis; here we investigate interstitial flow as a powerful morphoregulatory stimulant. We exposed both lymphatic and blood endothelial cells (LECs and BECs) to interstitial flow in 3D collagen gels as well as simple shear flow in 2D monolayers. We found that under interstitial flow (average 10 microm/s for 6 days), both cell types underwent drastic morphologic changes from static conditions: LECs formed large vacuoles and long extensions, while BECs formed multicellular branched lumen-containing networks. Under planar shear (20 dyn/cm2 for 24 h), LECs downregulated their cell-cell adhesions compared to BECs but did not differ morphologically; both aligned with flow as expected. The significance of these findings is twofold: first, they identify an important role of interstitial flow for in vitro microvascular organization and stabilization, and second, they demonstrate for the first time notable differences between LEC and BEC response to the biophysical environment, reflecting some of their functional differences in vivo.