Chromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies, resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98, and more than one half of these are homeobox transcription factors. By contrast, the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1), a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors, evidenced by a competitive growth advantage in liquid culture, increased replating efficient of colony-forming cells (CFCs), and a marked increase in spleen colony-forming cell output. Moreover, in a murine bone marrow transplantation model, NUP98-TOP1 expression led to a lethal, transplantable leukemia characterized by extremely high white cell counts, splenomegaly, and mild anemia. Strikingly, a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings, together with similar biologic effects reported for NUP98-HOX fusions, suggest unexpected, overlapping functions of NUP98 fusion genes, perhaps related to common DNA binding properties.