The development of stem cell-based neural repair strategies requires detailed knowledge on the interaction of migrating donor cells with the host brain environment. Here we report that overexpression of polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), in embryonic stem (ES) cell-derived glial precursors (ESGPs) strikingly modifies their migration behavior in response to guidance cues. ESGPs transduced with a retrovirus encoding the polysialyltransferase STX exhibit enhanced migration in monolayer cultures and an increased penetration of organotypic slice cultures. Chemotaxis assays show that overexpression of PSA results in an enhanced chemotactic migration toward gradients of a variety of chemoattractants, including fibroblast growth factor 2 (FGF2), platelet-derived growth factor, and brain-derived neurotrophic factor (BDNF), and that this effect is mediated via the phosphatidylinositol 3'-kinase (PI3K) pathway. Moreover, PSA-overexpressing ESGPs also exhibit an enhanced chemotactic response to tissue explants derived from different brain regions. The effect of polysialylation on directional migration is preserved in vivo. Upon transplantation into the adult striatum, PSA-overexpressing but not control cells display a targeted migration toward the subventricular zone. On the basis of these data, we propose that PSA plays a crucial role in modulating the ability of migrating precursor cells to respond to regional guidance cues within the brain tissue. Disclosure of potential conflicts of interest is found at the end of this article.