IGF-II is abundant in the nascent mesoderm of the gastrulating mouse embryo. Its function at this developmental stage is unknown. We investigated it by following the in vitro and in vivo differentiation of several androgenetic, biparental, parthenogenetic, and androgenetic Igf2 -/- murine ES cell lines; these cells differed in endogenous IGF-II levels because Igf2 is paternally expressed in the mouse embryo in most tissues. The expression of mesoderm markers and the subsequent formation of muscle structures were correlated with endogenous IGF-II level during teratoma formation and during in vitro differentiation. In addition, the absence of Igf2 in androgenetic Igf2 -/- ES cells led to a severe impairment of mesoderm development, demonstrating the dependence of the preferential mesoderm development of androgenetic ES cells upon Igf2 activity, among the numerous known imprinted genes. The addition of exogenous IGF-II to in vitro differentiation culture medium led to a specific increase in the expression of mesoderm markers. Thus, we propose a novel model in which the binding of IGF-II to its principal signaling receptor, IGF1R, at the surface of mesoderm precursor cells increases the formation of mesoderm cells.