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Background: Japanese Shiitake mushroom (Lentinula edodes) is known to be rich for nutrition, suggesting as the good source of bioactive compounds as well.  Objective: This study aimed to obtain compounds with effects on differentiation of neural stem cells into astrocytes that play important roles in the maintenance of neural functions. Materials and Methods: We examined the effects with 72 samples of food extracts on differentiation of neural stem cells. The assay was conducted using mouse embryonic stem cells (mESCs) introduced with the gene for the fusion protein of the differentiation marker of astrocyte (Glial Fibrillary Acidic Protein; GFAP) conjugated with the red fluorescent protein (mCherry), which is expressed when they differentiated into astrocytes. Neural stem cells induced from the mESCs were cultivated with samples, and the red fluorescence of mCherry in the induced astrocytes was measured. The induction efficiency of astrocytes was calculated on the basis of the number of the mCherry positive cells. As the results of the screening, Shiitake extract exhibited remarkable activity inducing differentiation into astrocytes. Thus, we fractionated the Shiitake extract with ODS flash column chromatography followed by HPLC. Results: The active substance inducing differentiation into astrocytes was isolated and identified. Conclusion: Shiitake contains a compound inducing astrocyte differentiation. Shiitake is expected to be useful for the functional food to prevent neurodegenerative diseases.