Members of the GATA and RUNX families of genes appear to have conserved functions during hematopoiesis from Drosophila to mammals. In Drosophila, the GATA factor Serpent (Srp) is required in blood cell progenitors for the formation of the two populations of blood cells (plasmatocytes and crystal cells), while the RUNX factor Lozenge (Lz) is specifically required for crystal cell development. Here we investigate the function and the mechanisms of action of Lz during hematopoiesis. Our results indicate that Lz can trigger crystal cell development. Interestingly, we show that Lz function is strictly dependent on the presence of functional Srp and that Srp and Lz cooperate to induce crystal cell differentiation in vivo. Furthermore, we show that Srp and Lz directly interact in vitro and that this interaction is conserved between Drosophila and mammals. Moreover, both Srp and mouse GATA1 synergize with mouse RUNX1 to activate transcription. We propose that interaction and cooperation between GATA and RUNX factors may play an important role in regulating blood cell formation from Drosophila to mammals.