Fyn, a member of the Src-family protein tyrosine kinase (PTK), is an essential factor in myelination in the CNS and is involved in murine embryonic stem (ES) cell growth and differentiation. Although dysfunctions of Fyn have been comparatively studied, the gain of function by ectopic expression, especially using ES cells, has seldom been investigated. In this article, we give the first report of the involvement of Fyn alteration in the sphere formation ability of murine ES cells. First, transient transfection of Fyn hardly affected multiplication and specialization. Then, we investigated Fyn overexpression using ES cells, which stably express Fyn. As a result, altered sphere formation capability was observed in all clones stably expressing Fyn. These results may provide important information for reproduction medical treatment using ES cells.