Crosstalk between endothelial cells (ECs) and pericytes or vascular smooth muscle cells (VSMCs) is essential for the proper functioning of blood vessels. This balance is disrupted in several vascular diseases but there are few experimental models which recapitulate this vascular cell dialogue in humans. Here, we developed a robust multi-cell type 3D vessel-on-chip (VoC) model based entirely on human induced pluripotent stem cells (hiPSCs). Within a fibrin hydrogel microenvironment, the hiPSC-derived vascular cells self-organized to form stable microvascular networks reproducibly, in which the vessels were lumenized and functional, responding as expected to vasoactive stimulation. Vascular organization and intracellular Ca²⁺ release kinetics in VSMCs could be quantified using automated image analysis based on open-source software CellProfiler and ImageJ on widefield or confocal images, setting the stage for use of the platform to study vascular (patho)physiology and therapy.