In the course of screening for cell differentiation inducers from botanical sources, the methanol extract of Withania somnifera L. (DUN.) showed activity. From the aerial parts of the plant, sixteen withanolides, including three new compounds, 14, 15 and 16, were isolated and their structures elucidated to be (20S, 22R)-4β, 5β, 6α, 27-tetrahydroxy-1-oxo-witha-2, 24-dienolide, (20R, 22R, 24S, 25R)-4β, 20β-dihydroxy-5β, 6β-epoxy-3β-methoxy-1-oxo-withanolide and 3-O-[β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl]-(20S, 22R)-1α, 3β-dihydroxywitha-5, 24-di-enolide, respectively, from the spectral data. Of these withanolides, 1, 2, 3 and 4 showed potent cell differentiation inducing activity against M1 cells. The most potent compound, 3, showed more potent activity than dexamethasone, the positive control. These active compounds have the same partial structure of the AB ring part, having a 4β-hydroxy-5β, 6β-epoxy-2-en-1-one moiety.