NPC differentiation is sensitive to EC phenotype (static or dynamic) and spatiotemporal placement. EC monolayers cultured on a transwell membrane were subjected to static (a, b) or dynamic (c, d) culture, while NPCs were cultured below at a distance of ~ 10 μm (a, c) or ~ 1000 μm (b, d) from the ECs. ECs were removed and NPCs were differentiated for 7 days then stained for neurons (NeuN+: green), astrocytes (GFAP+: yellow), or oligodendrocytes (O4+: red). (e) A significant increase (p < 0.1) in neurons far from dynamic EC cultures was observed compared to cells cultured in static cultures at any distance from static ECs, while cells near dynamic ECs demonstrate an increase in neurons, however, this is not significant compared to static paradigms. NPCs within 10 μm from static EC cultures exhibited a significant increase in oligodendrocytes compared to NPCs cultured in dynamic EC cultures. Data are represented as mean ± standard deviation. 50 μm scale bar.