We developed a multi-layer microfluidic platform for neural stem cells (NSCs)-derived neuronal differentiation. To culture NSCs in the multi-layer gradient microfluidic platform, we analyzed the molecular diffusion across the membrane using computational fluidic dynamic modeling and FITC-dextran fluorescence dye experiment. It showed that the membrane helped to generate the gradient in the multi-layer microfluidic platform. We also demonstrated that uniform-sized neurospheres docked inside the micropillar arrays differentiated into neuron cells for 6 days in the multi-layer microfluidic device. Furthermore, we analyzed the effect of drugs on neuronal cell fates in the multi-layer microfluidic platform. Therefore, this multi-layer microfluidic platform could be a potentially powerful tool to study neuronal differentiation and drug screening for neurodegenerative disease applications.