Physiologic chemoattractant gradients are shaped by diffusion, advection, binding to an extracellular matrix, and removal by cells. Previous in vitro tools for studying these gradients and the cellular migratory response have required cells to be constrained to a 2D substrate or embedded in a gel devoid of fluid flow. Cell migration in fluid flow has been quantified in the absence of chemoattractant gradients and shown to be responsive to them, but there is a need for tools to investigate the synergistic, or antagonistic, effects of gradients and flow. We present a microfluidic chip in which we generated precisely controlled gradients of the chemokine CCL19 under advective-diffusive conditions. Using torque-actuated membranes situated between a gel region and the chip outlet, the resistance of fluid channels adjacent to the gel region could be modified, creating a controllable pressure difference across the gel at a resolution inferior to 10 Pa. Constant supply and removal of chemokine on either side of the chip facilitated the formation of stable gradients at Péclet numbers between −10 and +10 in a collagen type I hydrogel. The resulting interstitial flow was steady within 0.05 μm s−1 for at least 8 h and varied by less than 0.05 μm s−1 along the gel region. This method advances the physiologic relevance of the study of the formation and maintenance of molecular gradients and cell migration, which will improve the understanding of in vivo observations.