The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions that drive lymph flow. Even though the signaling between the two main cellular components of a lymphangion, endothelial cells (LECs) and muscle cells (LMCs), is responsible for crucial lymphatic functions, there are no in vitro models that have included both cell types. Here, a fabrication technique (gravitational lumen patterning or GLP) is developed to create a lymphangion-chip. This organ-on-chip consists of co-culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells. The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy. In this device, lymphatic muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past. This system successfully characterizes the dynamics of cell size, density, growth, alignment, and intercellular gap due to co-culture and shear. Finally, exposure to pro-inflammatory cytokines reveals that the device could facilitate the regulation of endothelial barrier function through the lymphatic muscle cells. Therefore, this bioengineered platform is suitable for use in preclinical research of lymphatic and blood mechanobiology, inflammation, and translational outcomes.