Motor neuron diseases are neurodegenerative diseases that predominantly affect the neuromuscular system. To date, there are no valid therapeutic treatments for such diseases, and the classical experimental models fail in faithfully reproducing the pathological mechanisms behind them. In this regard, the use of three-dimensional (3D) culture systems, which more closely reproduce the native in vivo environment, can be a promising approach. Hydrogel-based systems are among the most used materials to reproduce the extracellular matrix, featuring an intrinsic similarity with its physiological characteristics. In this study, we developed a thermosensitive chitosan-based hydrogel combined with β-glycerophosphate (βGP) and sodium hydrogen carbonate (SHC), which give the system optimal mechanical properties and injectability, inducing the hydrogel sol-gel transition at 37 °C. An ad hoc protocol for the preparation of the hydrogel was established in order to obtain a highly homogeneous system, leading to reproducible physicochemical characteristics and easy cell encapsulation. All formulations supported the viability of a neuroblastoma/spinal cord hybrid cell line (NSC-34) beyond two weeks of culture and enabled cell differentiation towards a motor neuron-like morphology, characterized by the presence of extended neurites. Based on our results, these hydrogels represent excellent candidates for establishing 3D in vitro models of motor neuron diseases.