Recent reports indicate that neural stem cells (NSCs) exist in a cluster-like formation in close proximity to cerebral microvessels. Similar appearing clusters can be seen ex vivo in NSC cultures termed neurospheres. It is known that this neurosphere configuration is important for preserving stemness and a proliferative state. How NSCs form neurospheres or neuroclusters remains largely undetermined. In this study, we show that primary human NSCs express the tight junction proteins (TJPs): zonula occludens-1 (ZO-1), occludin, claudin-1, -3, -5, and -12. The relative mRNA expression was measured by quantitative polymerase chain reaction, and protein expression was confirmed by flow cytometry and immunofluorescence microscopy. Our results show that downregulation of TJPs occurs as neuronal differentiation is induced, suggesting that control of TJPs may be tied to the neuronal differentiation program. Importantly, upon specific knockdown of the accessory TJP, ZO-1, undifferentiated NSCs showed decreased levels of key stem cell markers. Taken together, our results indicate that TJPs possibly aid in maintaining the intercellular configuration of NSCs and that reduction in TJP expression consequently affects the stemness status.