Homeobox genes encode for regulatory proteins central to hematopoietic differentiation and proliferation. Previously, we 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-DeltaH3). Here, we demonstrate that this mutation results in a significantly truncated protein with impaired DNA-binding efficiency. Electrophoretic mobility shift assays (EMSA) confirm that wild-type HOXA11 (HOXA11-WT) interacts in vitro with the DNA-binding consensus sequence for HOXA11, and that this interaction is most efficient when the TALE transcription factor, Meis1b, is also present. However, the binding between HOXA11-DeltaH3 and DNA is abrogated even in the presence of Meis1b, suggesting the point mutant causes a disruption in the DNA-binding capacity. We investigated whether the point mutation also affected the physical protein-protein interaction between HoxA11 and Meis1b. Using GST pulldown assays, we find Meis1b interactions with both HOXA11-WT and HOXA11-DeltaH3 in the presence of DNA. DNAse treatment decreased these interactions, suggesting that the interaction is a protein-protein association, and DNA may serve to stabilize this interaction. Stable expression of FLAG-HOXA11-WT or -DeltaH3 in K562 cells significantly impacts megakaryocytic differentiation. Staurosporine (STSP) induced K562 cells to differentiate into a megakaryocytic phenotype. Treatment leads to an increase in surface expression of the megakaryocytic/platelet-specific antigen, CD61, and causes morphological changes consistent with megakaryocytic differentiation. CD61 surface expression on STSP treated HOXA11-WT and -DeltaH3 expressing cells was significantly reduced as compared to untransfected K562 cells. Interestingly, we found only a slight difference in CD61 expression between wild-type and mutant HOXA11 K562. These data suggest that HoxA11 inhibition of differentiation may involve nonhomeodomain sequences. Finally, our laboratory has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood and murine ES cell culture cocultured on OP9 for 6 days in the absence of leukemia inhibitory factor (LIF). This finding suggests HoxA11 may be endogenously expressed in very early hematopoietic precursor cells. Taken together, these data begin to give us insight into the molecular mechanisms by which HoxA11 may be involved in regulating megakaryocytic differentiation.