In vivo studies have suggested that gradients of CXCL12 (aka stromal cell-derived factor 1α) may be critical for neural stem cell (NSC) migration during brain development and neural tissue regeneration. However, traditional in vitro chemotaxis tools are limited by unstable concentration gradients and the inability to decouple cell migration directionality and speed. These limitations have restricted the reproducible and quantitative analysis of neuronal migration, which is required for mechanism-based studies. Using a microfluidic gradient generator, nestin and Sox-2 positive human embryonic NSC chemotaxis is quantified within a linear and stable CXCL12 gradient. While untreated NSCs are not able to chemotax within CXCL12 gradients, pre-treatment of the cells with brain-derived neurotrophic factor (BDNF) results in significant chemotactic, directional migration. BDNF pre-treatment has no effect on cell migration speed, which averages about 1 μm min−1. Quantitative analysis determines that CXCL12 concentrations above 9.0 nM are above the minimum activation threshold, while concentrations below 14.7 nM are below the saturation threshold. Interestingly, although inhibitor studies with AMD 3100 revealed that CXCL12 chemotaxis requires receptor CXCR4 activation, BDNF pre-treatment is found to have no profound effects on the mRNA levels or surface presentation of CXCR4 or the putative CXCR7 scavenger receptor. The microfluidic study of NSC migration within stable chemokine concentration profiles provides quantitative analysis as well as new insight into the migratory mechanism underlying BDNF-induced chemotaxis towards CXCL12.