The runt box (Runx) is a highly conserved DNA binding and protein-protein interaction domain that defines a family of heterodimeric transcription factors with essential roles in metazoan development. The first member of this family to be identified was the Drosophila regulatory gene runt, which was named by virtue of its function in establishing segmentation patterns during embryogenesis, and subsequently discovered to have additional functions in sex determination and neurogenesis. A second Drosophila Runx gene, lozenge, is required for cell patterning in the eye and for hematopoiesis. The genome project has revealed the existence of two additional Drosophila Runx genes, which to date have not been functionally characterized. Other invertebrate species with well-characterized Runx transcription factors include the nematode Caenorhabditis elegans and the sea urchin Strongylocentrotus purpuratus, each of which apparently contains only a single Runx gene. There are three Runx genes in mammals; Runx1 is required for definitive hematopoiesis and is a frequently mutated gene in human leukemia, Runx2 is required for osteogenesis and is associated with cleidocranial dysplasia, and Runx3 controls neurogenesis in the dorsal root ganglia and cell proliferation in the gastric epithelium, and is frequently deleted or silenced in human gastric cancer. Studies using mammalian systems and sea urchins indicate that Runx proteins have essential functions in both cell proliferation and differentiation, and in mammals they are both proto-oncogenes and tumor suppressors. Thus, a central question concerning the cell biology of Runx proteins is how are the opposing functions of this class of transcription factors regulated during development? Here I review current knowledge of Runx protein structure, function and regulation, and outline directions for future research aimed at understanding how Runx protein function is modulated during the transition from cell proliferation to differentiation in animal development.