During development and continuing into adulthood, stem cells function as a reservoir of undifferentiated cell types, whose role is to support cell genesis in several tissues and organs. In the adult, they play an essential homeostatic role by replacing differentiated cells that are lost due to physiological turnover, injury, or disease. The discovery of such cells in the adult mammalian central nervous system (CNS), an organ traditionally thought to have little or no regenerative capacity, has opened the door to the possibility of designing innovative regenerative therapeutics, an unexpected concept in neurobiology 15 years ago. In 1992, to detect precursor cells in the adult brain, we employed a serum-free culture system whereby the majority of primary differentiated CNS cells did not survive but a small population of EGF-responsive cells were maintained in an undifferentiated state and proliferated to form clusters, called neurospheres (Reynolds and Weiss, Science 255:1707-1710, 1992). These neurospheres could be (a) dissociated to form numerous secondary spheres or (b) induced to differentiate, generating the three major cell types of the CNS. This chapter outlines the adult mammalian NSC culture methodology and provides technical details of the neurosphere assay to achieve reproducible cultures.