Although the peripheral nervous system exhibits a higher rate of regeneration than that of the central nervous system through a spontaneous regeneration after injury, the functional recovery is fairly infrequent and misdirected. Thus, the development of successful methods to guide neuronal outgrowth, in vitro, is of great importance. In this study, a precise flow controlled microfluidic system with specific custom-designed chambers, incorporating laser-microstructured polyethylene terephthalate (PET) substrates comprising microgrooves, was fabricated to assess the combined effect of shear stress and topography on Schwann cells' behavior. The microgrooves were positioned either parallel or perpendicular to the direction of the flow inside the chambers. Additionally, the cell culture results were combined with computational flow simulations to calculate accurately the shear stress values. Our results demonstrated that wall shear stress gradients may be acting either synergistically or antagonistically depending on the substrate groove orientation relative to the flow direction. The ability to control cell alignment in vitro could potentially be used in the fields of neural tissue engineering and regenerative medicine.