ZO-1, ZO-2 and ZO-3 are closely related scaffolding proteins that link tight junction (TJ) transmembrane proteins such as occludin, claudins and junctional adhesion molecules to the actin cytoskeleton. Despite being among the first TJ proteins to have been identified and having undergone extensive biochemical analysis, little is know about the physiological roles of individual ZO proteins in different tissues or during vertebrate development. Here, we show that ZO-3-/- mice lack an obvious phenotype. In contrast, embryos deficient for ZO-2 die shortly after implantation due to an arrest in early gastrulation. ZO-2-/- embryos show decreased proliferation at E6.5, increased apoptosis at E7.5 and altered architecture of the apical junctional complex as compared to wild-type. ZO-1-/- mice are currently unavailable, while chimeric mice derived from ZO-1-/- embryonic stem (ES) cells are embryonic lethal. Because of the embryonic lethality of the ZO-1-/- and ZO-2-/- mice, we also generated knockout ES cell lines. We have obtained ZO-1-/-, ZO-2-/-, ZO-3-/-, ZO-1-/-ZO-2-/-, and ZO-2-/-ZO-3-/- ES cells. These cell lines have shown various defects in their ability to differentiate into epithelial cells, cardiomyocytes or skeletal muscle cells.