Hematopoietic stem cells (HSCs) are unique in their capacity to maintain blood formation following transplantation into immunocompromised hosts. Expansion of HSCs in vitro is therefore important for many clinical applications but has met with limited success because the mechanisms regulating the self-renewal process are poorly defined. We have previously shown that expression of the LIM-homeobox gene Lhx2 in hematopoietic progenitor cells derived from embryonic stem cells differentiated in vitro generates immortalized multipotent hematopoietic progenitor cell lines. However, HSCs of early embryonic origin, including those derived from differentiated embryonic stem cells, are inefficient in engrafting adult recipients upon transplantation. To address whether Lhx2 can immortalize hematopoietic progenitor/stem cells that can engraft adult recipients, we expressed Lhx2 in hematopoietic progenitor/stem cells derived from adult bone marrow. This approach allowed for the generation of immortalized growth factor-dependent hematopoietic progenitor/stem cell lines that can generate erythroid, myeloid, and lymphoid cells upon transplantation into lethally irradiated mice. When transplanted into stem cell-deficient mice, these cell lines can generate a significant proportion of circulating erythrocytes in primary, secondary, and tertiary recipients for at least 18 months. Thus, Lhx2 immortalizes multipotent hematopoietic progenitor/stem cells that can generate functional progeny following transplantation into lethally irradiated hosts and can long-term repopulate stem cell-deficient hosts.