The epigenetic signals and progenitor cell species involved in progressive neural maturation in the mammalian brain are poorly understood. Although these complex developmental issues can be examined in cultures of generative zone progenitor cells, analysis of signaling relationships in complex progenitor cell systems requires the meticulous definition of the cellular complement at each developmental stage. The presence of microglia within the generative zone cultures would further complicate these developmental analyses. Utilizing the microglial markers Griffonia simplicifolia B4 isolectin, carbocyanine dye-acetylated low density lipoprotein, F4/80, and Mac-1 we now report the presence of microglia within cultures of late embryonic murine epidermal growth factor-derived generative zone progenitor cells. Cytokine treatment of serially passaged epidermal growth factor-generated neurospheres altered the phenotype of the microglia in culture. Macrophage colony-stimulating factor treatment promoted the expression of spindle-shaped microglia, whereas granulocytemacrophage colony-stimulating factor treatment promoted the elaboration of flat and amoeboid microglia. Treatment with microglial-conditioned medium or 10% non-heat inactivated fetal calf serum led to an increased complement of both phenotypes. Microglia could be generated from single isolated neurospheres, and there were differences in the number of microglial lineage species obtained from distinct oligopotent progenitor cells, raising the possibility that a complement of this cellular lineage may be derived from a progenitor cell present within the generative zones. These observations indicate that microglia are present within the generative zone progenitor cell system, and this system thus represents an important experimental resource to examine the progenitor cell maturation and the origin of the microglial lineage.