Eph proteins are receptors with tyrosine-kinase activity which, with their ephrin ligands, mediate contact-dependent cell interactions that are implicated in the repulsion mechanisms that guide migrating cells and neuronal growth cones to specific destinations. Ephrin-B proteins have conserved cytoplasmic tyrosine residues that are phosphorylated upon interaction with an EphB receptor, and may transduce signals that regulate a cellular response. Because Eph receptors and ephrins have complementary expression in many tissues during embryogenesis, bidirectional activation of Eph receptors and ephrin-B proteins could occur at interfaces of their expression domains, for example at segment boundaries in the vertebrate hindbrain. Previous work has implicated Eph receptors and ephrin-B proteins in the restriction of cell intermingling between hindbrain segments. We therefore analysed whether complementary expression of Eph receptors and ephrins restricts cell intermingling, and whether this requires bidirectional or unidirectional signalling. Here we report that bidirectional but not unidirectional signalling restricts the intermingling of adjacent cell populations, whereas unidirectional activation is sufficient to restrict cell communication through gap junctions. These results reveal that Eph receptors and ephrins regulate two aspects of cell behaviour that can stabilize a distinct identity of adjacent cell populations.