The preimplantation development of the mouse embryo leads to the formation of two populations of cells: the trophectoderm, which is a perfect epithelium, and the inner cell mass. The divergence between these two lineages is the result of asymmetric divisions, which can occur after blastomere polarization at compaction. The apical pole of microvilli is the only asymmetric feature maintained during mitosis and polarity is reestablished only in daughter cells that inherit all or a sufficient part of this pole. To analyze the role of ezrin in the formation and stabilization of the pole of microvilli, we isolated and cultured inner cell masses (ICM). These undifferentiated cells can differentiate very quickly into epithelial cells. After isolation of the ICMs, ezrin relocalizes at the cell cortex before the formation of microvilli. This redistribution occurs in the absence of protein synthesis. The formation of microvilli at the apical surface of the outer cells of ICM correlates with a major posttranslational modification of ezrin. We show here that this posttranslational modification is not controlled by a serine/threonine kinase but an O-glycosylation may partially contribute to it. These data suggest that ezrin has at least two roles during development. First, ezrin may be involved in the formation of microvilli because it localizes at the cell cortex before microvilli appear in ICMs. Second, ezrin may stabilize the pole of microvilli because it is modified posttranslationally when microvilli form.