Abstract Homeobox genes encode for regulatory proteins central to bone morphogenesis as well as hematopoietic differentiation and proliferation. Previously we have identified an inherited syndrome of congenital amegakaryocytic thrombocytopenia and radio-ulnar synostosis that is associated with a point mutation in the third helix of HOXA11 homeodomain (HOXA11-ΔH3). We postulated that this mutation results in a truncated protein with impaired DNA binding efficiency. Compared to wild type (HOXA11-WT) in vitro transcription and translation of HOXA11-ΔH3 produced a truncated form of the protein. Electropheretic mobility shift assays (EMSA) confirmed that product from a HoxA11-WT cDNA expression plasmid interacts with the DNA binding consensus sequence for HoxA11 and this interaction is most efficient when its TALE transcription co-factor, Meis1b, is also present. Using the truncated HOXA11-ΔH3 product, the mobility shift was abrogated even in the presence of Meis1b, suggesting the point mutant causes a disruption in the DNA binding capacity of HoxA11. We investigated whether the point mutation affected the physical protein-protein interaction that occurs between HoxA11 and Meis1b. Using GST-pulldown assays we examined these interactions. Meis1b showed a 11.8±4.3 fold increase in interaction with HOXA11-WT and a 13.7±3.6 fold increase with HOXA11-ΔH3 in the presence of DNA. DNAse treatment decreased the interactions to 6±0.24 and 7±1.1 for HOXA11-WT and -ΔH3 respectively. These data suggest the physical interaction between HoxA11 and Meis1b is a protein-protein association and that the presence of DNA may serve to stabilize this interaction. We next investigated the effects of HOXA11 on megakaryocytic differentiation. The human erythroleukemia cell line, K562, was stably transfected with expression vectors for either HOXA11-WT or -ΔH3. The transfected cell lines were then treated with the pharmacological agents, phorbol-12 myristate-13 acetate (PMA) or staurosporine (STSP), to induce megakaryocytic differentiation. Both agents induce surface expression of the megakaryocytic/platelet-specific antigen, CD61 and cause morphological changes. CD61 expression in untransfected cells increased from 1.6±0.4% at baseline to 91±0.5% with PMA and to 81±2.4% when treated with STSP. Marked differences in CD61 expression and viability were seen in transfected cells. HOXA11-WT and -ΔH3 lead to a significantly less CD61 expression, 53±10% and 69.8±5.8% respectively following PMA induction, compared to untransfected cells. PMA treatment of cells that constitutively express HOXA11-WT or -ΔH3 resulted in a marked reduction in cell viability and suggests that the normal silencing of HOXA11 in early hematopoiesis may be an essential aspect of its function. Neither HOXA11-WT nor -ΔH3 cells showed an increase in expression of CD61 expression following STSP treatment, 10.1±5.9% and 3.7±2.4% respectively. Interestingly, nearly all transfected cells treated with STSP remained viable, suggesting divergent signaling pathways are utilized by this differentiating agent. There were no substantial differences between wild type and mutant HOXA11 with either inducing agent, suggesting that inhibition of differentiation by HOXA11 involves other non-homeodomain regulatory sequences of this gene. Taken together these data begin to give us insight into the molecular mechanisms by which HoxA11 regulates megakaryocytic differentiation.