There is a demand for three-dimensional (3D) angiogenesis model including endothelial cells (ECs) and mesenchymal stem cells (MSCs), which are known to differentiate into pericytes, to construct stabilized and matured microvascular networks in vitro. However, it remains to be elucidated how MSCs affected on ECs in the process of 3D angiogenesis. In this study, we utilized a microfluidic device to develop a 3D coculture system including human umbilical vein ECs and human MSCs, which allowed us to investigate the effects of MSCs on ECs in the context of 3D angiogenesis. A series of EC:MSC ratio was tested in the EC-MSC coculture. First, we confirmed that MSCs differentiated into pericytes by direct EC-MSC contacts. Next, we found that MSCs attenuated vascular sprout formation of ECs regardless of EC:MSC ratio in the early stage of 3D angiogenesis as well as extension of microvascular networks in the later stage. ECs and MSCs were also cultured under interstitial flow to enhance angiogenesis. However, the stabilization effects of MSCs on the extension of capillary structures were dominant over the promotion effects of the interstitial flow. These results indicate the stabilization effect of MSCs on the formation of microvascular networks in vitro. Although some HMSCs differentiated into pericytes and located around microvascular networks, vascular structures became thick over time in coculture. The 3D EC-MSC coculture model described in this study is useful to further investigate culture microenvironments for constructing stabilized and matured microvascular networks with aligning pericytes.