Decreased interstitial flow (IF) in secondary lymphedema is coincident with poor physiological lymphatic regeneration. However, both the existence and direction of causality between IF and lymphangiogenesis remain unclear. This is primarily because the role of IF and its importance relative to the action of the prolymphangiogenic growth factor vascular endothelial growth factor (VEGF)-C (which signals primarily through its receptor VEGFR-3) are poorly understood. To clarify this, we explored the cooperative roles of VEGFR-3 and IF in a mouse model of lymphangiogenesis in regenerating skin. Specifically, a region of lymphangiogenesis was created by substituting a portion of mouse tail skin with a collagen gel within which lymphatic capillaries completely regenerate over a period of 60 days. The relative importance of IF and VEGF-C signaling were evaluated by either inhibiting VEGFR-3 signaling with antagonistic antibodies or by reducing IF. In some cases, VEGF-C signaling was then increased with exogenous protein. To clarify the role of IF, the distribution of endogenous matrix metalloproteinases (MMPs) and VEGF-C within the regenerating region was determined. It was found that inhibition of either VEGFR-3 or IF suppressed endogenous lymphangiogenesis. Reduction of IF was found to decrease lymphatic migration and transport of endogenous MMP and VEGF-C through the regenerating region. Therapeutic VEGF-C administration restored lymphangiogenesis following inhibition of VEGFR-3 but did not increase lymphangiogenesis following inhibition of IF. These results identify IF as an important regulator of the pro-lymphangiogenic action of VEGF-C.