Interstitial fluid flow, critical for macromolecular transport, was recently shown to drive fibroblast differentiation and perpendicular cell and matrix alignment in 3D collagen cultures. Here we explore the mechanisms underlying this flow-induced cell and collagen alignment. Cell and matrix alignment was assessed from 3D confocal reflectance stacks using a Fast Fourier Transform method. We found that human dermal and lung fibroblasts align perpendicular to flow in the range of 5–13 μm/s (0.1–0.3 dyn/cm²) in collagen; however, neither cells nor matrix fibers align in fibrin cultures, which unlike collagen, is covalently cross-linked and generally degraded by cell fibrinolysis. We also found that even acellular collagen matrices align weakly upon exposure to flow. Matrix alignment begins within 12 h of flow onset and continues, along with cell alignment, over 48 h. Together, these data suggest that interstitial flow first induces collagen fiber alignment, providing contact guidance for the cells to orient along the aligned matrix; later, the aligned cells further remodel and align their surrounding matrix fibers. These findings help elucidate the effects of interstitial flow on cells in matrices and have relevance physiologically in tissue remodeling and in tissue engineering applications.