Development of the vertebrate central nervous system is characterized by significant long distance cell migration. While the radial migration of neuronal precursors to their final location is well established the migration of glial cells and their precursors is less well understood. To define the pathways of migration and dissect the cell and molecular mechanisms mediating such migration requires the development of appropriate models. Here we show that purified neonatal astrocytes injected into organotypic slice cultures of developing cerebral cortex migrate in defined patterns depending on where they are placed within the tissue. Injection into gray matter resulted in radially oriented migration either towards the pial or ventricular surface. By contrast injection into developing white matter resulted in largely longitudinal migration along developing axon tracts. While the cytoarchitecture of the tissue influenced the pattern of migration, the extent of migration appeared to be regulated primarily by the age of the host tissue. Homochronic injections performed prior to postnatal day 4 resulted in extensive migration while after day 7 migration was relatively limited. Heterochronic injections indicated that while astrocytes within the 1st postnatal week retained the capacity to migrate extensively, older tissue failed to support extensive migration of either young or old astrocytes. These data suggest the existence of distinct migrational cues in the CNS and that environmental, not cell intrinsic properties primarily regulate astrocyte migration through the developing cortex.