All-trans-retinoic acid (RA) stimulates differentiation of normal hematopoietic progenitors and acute myeloid leukemia cells. GATA-2 is a transcription factor expressed in early progenitor cells and implicated in the control of the fate of hematopoietic stem cells and progenitor cells. We have investigated the possibility that the GATA and nuclear hormone receptor pathways are functionally linked through direct protein-protein interaction. Here we demonstrate that in human myeloid KG1 cells, RA receptor alpha (RARα), the major RAR expressed in hematopoietic cells, associates with GATA-2. This association is mediated by the zinc fingers of GATA-2 and the DNA-binding domain of RARα. As a consequence of this interaction, RARα is tethered to the DNA sites that are recognized and bound by GATA-2, and the transcriptional activity of GATA-2 becomes RA responsive. The RA responsiveness of GATA-dependent transcription is eliminated by expression of either a dominant negative form of RARα or a GATA-2 mutant that fails to interact with RARα. Overexpression of RXRα inhibits RARα binding to the GATA-2-DNA complex, thus resulting in attenuation of the effects of RARα on GATA-2 activity. In addition, inhibition by RA of GATA-2-dependent hematopoietic colony formation in an embryonic stem cell model of hematopoietic differentiation provided biological evidence for functional cross talk between RA and GATA-2-dependent pathways.