Protocols for culturing neural stem cells (NSCs) are increasingly finding utilization for studying and growing of tissues that can appropriately model the neural regeneration processes. Two-dimensional (2D) plastic or glass surface-enabled mammalian cell cultures have been the platforms for performing in vitro cell cultures. Isolated mammalian cells, however, come from three-dimensional (3D) spaces, thus recapitulating such 3D microenvironments is among the challenges for many tissue engineering applications. Herein, we present the protocols for culturing NSCs in 3D polysaccharide-based hydrogel microenvironments that mimic, for instance, the native extracellular matrix (ECM) space (of the brain). The protocols include three key steps: (1) generation of 3D hydrogels with living cells, (2) culturing NSCs in 3D environments, and (3) characterization via immunostaining and genetic expression assay (RT-qPCR).