The standard culture method for neural stem cells cannot prevent the attachment of neurospheres, which eventually result in differentiation. This study developed a new method for long-term neural stem cell cultivation. In the antiattachment group, neural stem cells were cultured in flasks coated with 1.5% agarose gel. As a control, cells were cultured in plastic flasks. The 5-bromine-deoxyuridine incorporation assay was used to determine the S-phase labeling index of both groups. The methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to determine the total cell vitality. After a 3-mo culture, the spontaneous differentiation of stem cells was studied using immunocytochemistry for neuroepithelial stem cell protein. We found that neural stem cells grew rapidly in the antiattachment flasks. There was no statistically significant difference between the two groups in terms of the S-phase labeling index or MTT assay. When cultured for 3 mo in vitro, many more cells differentiated in the control than in the antiattachment group (32.05 vs. 0.64%, P<0.01). Moreover, the neural stem cells in the antiattachment group remained multipotent. Therefore, flasks coated with agarose gel are suitable for long-term neural stem cell culture.