The influence of astrocytes on the development of cerebral cortical neurons was studied in a co-culture system using transwells with chemically defined medium. Cerebral cortical neurons from 15- or 16-day-old mouse embryo were cultured in the lower wells which were separated by a porous membrane from the upper transwells where cerebral cortical astrocytes from newborn mouse were cultured. Neurons co-cultured with astrocytes for 7 days formed a network-like web and maintained a slightly better survival from 4 to 7 days. However, neurons cultured in conditioned medium obtained from astrocytes did not form any network after 7 days even though they maintained a better cell survival at 4 days.