GATA-1, the founding member of GATA family, is a master gene for erythropoiesis. It binds to the consensus sequence WGATAR and is involved in the activation of most of the erythroid specific genes. GATA-1 can also repress the transcription of genes coding for proteins involved in the control of cell proliferation. Here we describe a novel function of GATA-1 i.e. the control of the cell proliferation through a direct association to the retinoblastoma protein (pRb). This association required the canonical LXCXE motif localized between amino acids 81 and 85 of GATA-1. This sequence is conserved during evolution, is not found in the other members of the GATA family and is absent in the short form of GATA-1 expressed in the acute megakaryoblastic leukemia of Down syndrome. The inhibition of cellular proliferation induced by GATA-1 over-expression was inefficient in pRb negative cell lines or when pRb was knocked down by specific shRNA and, using a GATA-1 protein mutated on the LXCXE motif (GATA-1Rb-), we showed that (i) the LXCXE motif was necessary for GATA-1/pRb association, (ii) the over-expression of GATA-1Rb- did not inhibit cellular proliferation whereas GATA-1 over-expression did, and (iii) the expression of GATA-1Rb- in a GATA-1 deficient erythroid cell line decreased the number of benzidine positive cells and increased the immature erythroid fraction. Finally, endogenous level expression of GATA-1Rb- with GATA-1 HRD vector in the GATA-1.05 genotype mice resulted in a partial rescue of GATA-1.05 embryos from the early lethality at E11.5, but the embryos then exhibited severe anemia and died by E15.5. Analysis of the E12.5 fetal liver of these GATA-1Rb- rescued mice showed an increased number of immature erythroid cells and a decreased number of mature erythroid cells. These results indicate that the GATA-1/pRB interaction is necessary for erythropoiesis enlighting the mechanism of the autonomous defect of erythropoiesis observed in the Rb−/− mice.