For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP⁺CD45.2⁺ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP⁺ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10³-fold more MAPCs were required for efficient engraftment. Because GFP⁺ host-derived CD45.1⁺ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.