During development and pathological processes, cells in the central nervous system (CNS) are highly motile. Despite the fact that cell motion is driven by forces, our current understanding of the physical interactions between CNS cells and their environment is very limited. We here show how nanometer deformations of CNS tissue caused by piconewton forces exerted by cells contribute to regulating CNS development and pathologies. In vitro, growth and migration velocities, directionality, cellular forces as well as neuronal fasciculation and maturation all significantly depended on substrate stiffness. Moreover, when grown on substrates incorporating linear stiffness gradients, glial cells migrated towards stiffer, while axon bundles turned towards softer substrates. In vivo time-lapse atomic force microscopy revealed stiffness gradients in developing brain tissue, which axons followed as well towards soft. Interfering with brain stiffness and mechanosensitive ion channels in vivo both led to similar aberrant neuronal growth patterns with reduced fasciculation and pathfinding errors. Importantly, CNS tissue significantly softened after traumatic injuries. Ultimately, mechanical signals not only directly impacted neuronal growth but also indirectly by regulating neuronal responses to and the availability of chemical guidance cues, strongly suggesting that chemical and mechanical signaling pathways are intimately linked, and that their interaction is crucial for neuronal development and regeneration.