Adult neurogenesis in the hippocampus is known to enhance pattern separation. However, the effect of adult neurogenesis on spatial pattern separation at the cellular assembly level is unclear. In order to elucidate how newborn and immature neurons change learning of spatial pattern of mature neuronal network, we evaluated evoked response to two types of spatial patterns of the cultured hippocampal network with or without added neural stem cells by using electrical stimulation on microelectrode array. Results show that the existence of newborn and immature neurons changed evoked response of mature neuronal network to both trained and untrained patterns, suggesting that the presence of immature neurons may contribute to production of the change that mature neuronal network enhances LTP and excitation to stimuli.