The neurosphere assay is the gold standard for assessing the proliferative and differentiation capacities of neural progenitor cells (NPCs). Here, we present a protocol for isolating, propagating, and maintaining hippocampal neurospheres from adult and aged mice and differentiating cultured NPCs into neurons and astrocytes. We describe steps for establishing a heterochronic co-culture of neurosphere-derived cells with primary neurons. Using neurospheres from old animals enables investigation of the effects of aging on the development and differentiation of newborn neurons.