A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax‐related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t (11; 19)(q23; p13. 3) translocation express HRX–ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor (s). To characterize directly the potential transforming effects of HRX–ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin‐3 and stem cell factor. Immortalized cells contained structurally intact HRX–ENL proviral DNA and expressed a low‐level of HRX–ENL mRNA. In contrast, wild‐type ENL or a deletion mutant of HRX–ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX–ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX–ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain‐of‐function mechanism for HRX–ENL‐mediated leukemogenesis.