Interstitial flow in articular cartilage is secondary to compressive and shear deformations during joint motion and has been linked with the well-characterized heterogeneity in structure and composition of its extracellular matrix. In this study, we investigated the effects of introducing gradients of interstitial flow on the evolution of compositional heterogeneity in engineered cartilage. Using a parallel-plate bioreactor, we observed that Poiseuille flow stimulation of chondrocyte-seeded agarose hydrogels led to an increase in glycosaminoglycan and type II collagen deposition in the surface region of the hydrogel exposed to flow. Experimental measurements of the interstitial flow fields based on the fluorescence recovery after photobleaching technique suggested that the observed heterogeneity in composition is associated with gradients in interstitial flow in a boundary layer at the hydrogel surface. Interestingly, the interstitial flow velocity profiles were nonlinearly influenced by flow rate, which upon closer examination led us to the original observation that the apparent hydrogel permeability decreased exponentially with increased interfacial shear stress. We also observed that interstitial flow enhances convective mass transport irrespective of molecular size within the boundary layer near the hydrogel surface and that the convective contribution to transport diminishes with depth in association with interstitial flow gradients. The implications of the nonlinearly inverse relationship between the interfacial shear stress and the interstitial flux and permeability and its consequences for convective transport are important for tissue engineering, since porous scaffolds comprise networks of Poiseuille channels (pores) through which interstitial flow must navigate under mechanical stimulation or direct perfusion.