Remyelination of the central nervous system (CNS) is a regenerative response that depends on the development of oligodendrocyte precursor cells (OPCs), which are generated from neural stem cells in developmental stages and exist as tissue stem cells in the adult CNS. Three-dimensional (3D) culture systems that recapitulate the complexity of the in vivo microenvironment are important for understanding the behavior of OPCs in remyelination and for exploring effective therapeutic approaches. In general, functional analysis of OPCs has mainly used two-dimensional (2D) culture systems; however, the differences between the properties of OPCs cultured in 2D and 3D have not been fully elucidated despite cellular functions being affected by the scaffold. In this study, we analyzed the phenotypic and transcriptomic differences in OPCs from 2D and collagen gel-based 3D cultures. In the 3D culture, the OPCs exhibited less than half ratio of proliferation and almost half ratio of differentiation to mature oligodendrocytes, compared to the 2D culture in the same culturing period. RNA-seq data showed robust changes in the expression level of genes associated with oligodendrocyte differentiation, and there were more up-regulated genes than down-regulated genes in 3D cultures compared to 2D cultures. In addition, the OPCs cultured in collagen gel scaffolds at lower collagen fiber densities showed higher proliferation activity compared with those cultured in collagen gel with higher collagen fiber densities. Our findings have identified the effect of culture dimension as well as the complexity of the scaffold on OPC responses at the cellular and molecular levels.