A previous small molecule screen demonstrated that some prenylflavonoids can promote neuronal differentiation from mouse embryonic stem (ES) cells based on morphologic criteria. Here we build on this observation and examine the neuronal subtypes induced by icaritin, a compound screened, and the molecular events underlying the differentiation. In the presence of icaritin, the number of neural rosettes in embryoid bodies (EBs) expressing nestin efficiently increased and the neuroectodermal gene Fgf5 expression upregulated during germ layer formation. The neural progenitors generated from icaritin-treated EBs were further differentiated into the neurons (marked by beta-tubulin III) and also enhanced the choline acetyltransferase (ChAT) expression upon terminal differentiation. A suppression of p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and sustained extracellular signal-regulated protein kinase (ERK) phosphorylation existed simultaneously without estrogen-like activities involved. Taken together, enhanced co-expression of beta-tubulin III and choline acetyltransferase in neuronal differentiation from mouse ES cells is promoted by icaritin via estrogen receptor-independent action.