The 8;21 translocation is a common chromosomal abnormality in acute myeloid leukemia (AML). We recently identified a naturally occurring leukemogenic splice variant, AML1-ETO9a (acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor-9a), of t(8;21). To understand the leukemic potential of AML1-ETO9a, we performed microarray analysis with the murine multipotential hematopoietic FDCP-mix A4 cell line. We identified changes in expression of various genes including CD44. CD44 is a type I transmembrane protein and functions as the major cellular adhesion molecule for hyaluronic acid, a component of the extracellular matrix. CD44 is expressed in most human cell types and is implicated in myeloid leukemia pathogenesis. We show that the presence of AML1-ETO9a significantly increased the expression of CD44 at both RNA and protein levels. Furthermore, the CD44 promoter is bound by AML1-ETO9a and AML1-ETO at the chromatin level. In addition, in the AML1-ETO9a leukemia mouse model CD44 is regulated in a cell context-dependent manner. Thus, our observations suggest that AML1-ETO and its splice variant AML1-ETO9a are able to regulate the expression of the CD44 gene, linking the 8;21 translocation to the regulation of a cell adhesion molecule that is involved in the growth and maintenance of the AML blast/stem cells.