Brain tumor invasion leads to recurrence and resistance to treatment. Glioma cells invade in distinct patterns, possibly determined by microenvironmental cues including chemokines, structural heterogeneity, and fluid flow. We hypothesized that flow originating from pressure differentials between the brain and tumor is active in glioma invasion. Using in vitro models, we show that interstitial flow promotes cell invasion in multiple glioma cell lines. Flow effects were CXCR4-dependent, because they were abrogated by CXCR4 inhibition. Furthermore, CXCR4 was activated in response to flow, which could be responsible for enhanced cell motility. Flow was seen to enhance cell polarization in the flow direction, and this flow-induced polarization could be blocked by CXCR4 inhibition or CXCL12 oversaturation in the matrix. Furthermore, using live imaging techniques in a three-dimensional flow chamber, there were more cells migrating and more cells migrating in the direction of flow. This study shows that interstitial flow is an active regulator of glioma invasion. The new mechanisms of glioma invasion that we identify here-namely, interstitial flow-enhanced motility, activation of CXCR4, and CXCL12-driven autologous chemotaxis-are significant in therapy to prevent or treat brain cancer invasion. Current treatment strategies can lead to edema and altered flow in the brain, and one popular experimental treatment in clinical trials, convection enhanced delivery, involves enhancement of flow in and around the tumor. A better understanding of how interstitial flow at the tumor margin can alter chemokine distributions, cell motility, and directed invasion offers a better understanding of treatment failure. .