Purpose: To investigate whether conditioned medium from embryonic rat retinas can induce differentiation of adult rat hippocampus-derived neural stem cells (AHSCs) into neurons and glia in vitro. Methods: AHSCs were cultured in 3 types of media: standard culture medium, conditioned medium from embryonic rat retina, and standard culture medium with retinoic acid. Neuronal and glial differentiation of the cultured cells was assessed by cell growth analysis, flow cytometric analysis, immunofluorescent staining, and RT-PCR analysis. Results: Cells cultured in the standard medium showed very little neuronal and glial differentiation. The cells cultured in the conditioned medium and the medium with retinoic acid showed neuronal morphology and growth inhibition. They also expressed mature neuronal markers and glial markers. In addition, the cells cultured in the conditioned medium expressed Thy-1, HPC-1, and calbindin, which were not found in the previous studies with postnatal retinas in vivo. Those cultured in the medium with retinoic acid expressed HPC-1 and calbindin, but not Thy-1. Conclusions: Conditioned medium from embryonic rat retina contains factors that induce neuronal and glial cell differentiation of AHSCs, and promote up-regulation of some types of retinal cell markers.