The establishment and maintenance of precisely organized tissues requires the formation of sharp borders between distinct cell populations. The maintenance of segregated cell populations is also required for tissue homeostasis in the adult, and deficiencies in segregation underlie the metastatic spreading of tumor cells. Three classes of mechanisms that underlie cell segregation and border formation have been uncovered. The first involves differences in cadherin-mediated cell–cell adhesion that establishes interfacial tension at the border between distinct cell populations. A second mechanism involves the induction of actomyosin-mediated contraction by intercellular signaling, such that cortical tension is generated at the border. Third, activation of Eph receptors and ephrins can lead to both decreased adhesion by triggering cleavage of E-cadherin, and to repulsion of cells by regulation of the actin cytoskeleton, thus preventing intermingling between cell populations. These mechanisms play crucial roles at distinct boundaries during development, and alterations in cadherin or Eph/ephrin expression have been implicated in tumor metastasis.