Neural stem cells are multipotent and self-renewing cells with important potential application in cell replacement therapy in brain damage. Many studies have shown that nestin-positive cells represent neural stem and progenitor cells in the central neural system. Here, we derived neural stem cells from the subventricular zone of a newborn nestin-promoter-driven green fluorescent protein mouse, and found that the percentage of nestin-positive cells decreased continuously at each passage in neurosphere culture. Using the relative proliferation ratio and relative division ratio analysis, we concluded that the slower cycling of nestin-positive cells was responsible for this decrease.