Myelin sheaths, formed by oligodendrocytes (OLs), enable rapid signal transfer in central nervous system (CNS) and loss or damage of myelin sheaths results in many neurological disorders such as Alzheimer’s or multiple sclerosis. However, detailed mechanism of myelination process remains largely unknown, not only because of the complicated reciprocal signaling process but also the lack of in vitro model systems that allow easy experimental manipulations. We present a 3D microfluidic neural stem/progenitor cell (NSPC) culture platform (i.e., Brain-on-a-Chip) capable of culturing NSPCs in an aggregate form under spatially controlled microenvironment and expect it to be used as an in vitro model system for studying CNS myelinogenesis. The platform is composed of four culture chambers each containing 10 horseshoe shaped trapping sites. Finite element method (FEM) computer simulation was used to confirm a uniform media flow within the culture chamber and to optimize the design/dimension of the trapping structures for increased aggregate trapping efficiency. NSPC aggregates with uniform pre-determined size (150 µm) were prepared by culturing dissociated NSPCs from E16 rats in a micro-well array (depth: 150 µm, diameter: 150 µm) for 3 days. NSPC aggregates were collected and loaded into the culture platform where they were captured at each trapping sites with trapping efficiency of approximately 85%. Robust neurite outgrowth and glial migration were observed during the first week of culture. At DIV 14, the NSPC aggregates were treated with retinoic acid (500 nM) to investigate its effect on myelin formation in vitro. After two weeks of treatment, increased number of myelinating OLs and myelin segments were found in retinoic acid-treated group as compared to controls. In summary, we have developed a microfluidic NSPC culture platform that can be exploited as a powerful tool for investigating neural development and myelination in vitro and to test potential drug candidates capable of promoting CNS myelination.