We introduced mechanical stimuli and micropatterned substrate with microfibers to investigate their effects on neurite outgrowth along with nerve growth factor in vitro. Two types of surface morphology were used: a surface that was coated by laminin alone and a surface where in microfibers was added on the laminin surface. PC-12 cells were seeded on both surface types and cultured for 2 d. The magnitudes of shear stresses ranged from 0.10 to 1.50 Pa. Two days after stimulation by shear stress, neurite outgrowth and its direction were measured by F-actin staining and digital image processing. When a shear stress of 0.50 Pa was applied, neurons were most highly aligned with microfibers. The average length of neurite outgrowth with microfibers was largest at a shear stress of 0.25 Pa. The results suggest that micropatterned fibers and fluid-induced shear stress are promising for stimulating neurite outgrowth in a desired direction.