Anodic aluminum oxide (AAO) membranes were fabricated in a mild two-step anodization procedure. The voltage was varied during both anodization steps to control the pore size and morphology of the AAO membranes. Pore sizes ranged from 34 nm to 117 nm. Characterization of the pore structure was performed by scanning electron microscopy (SEM). To assess the potential of the AAO membranes as a neuronal differentiation platform, C17.2 neural stem cells (NSCs), an immortalized and multipotent cell line, were used. The NSCs were forced to differentiate via serum-withdrawal. Cellular growth was characterized by immunocytochemistry (ICC) and SEM. ImageJ software was used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal network density and neurite outgrowth length was achieved.