The immortalized murine stromal cell line AFT024 has been reported to maintain human hematopoietic progenitors in an undifferentiated state in vitro. In the current studies the beige/nude/xid (bnx) mouse in vivo xenograft model was used to examine the engraftment and multilineage generative potential of human hematopoietic progenitors after 2-3 weeks growth on AFT024 stroma, in comparison to primary stromal monolayers derived from post-natal human bone marrow. Eight to 12 months after transplantation of human CD34+CD38- cells from umbilical cord blood, cultured on AFT024 vs human stroma for 2-3 weeks, the murine bone marrow was harvested and analyzed for the presence of human myeloid and lymphoid cells. The mean percent engraftment of total human hematopoietic cells in the murine marrow was significantly higher after co-cultivation on AFT024 than on human stroma. Human myeloid and lymphoid lineage cells were detected in all mice. However, engraftment of myeloid lineage cells (CD33+), B lymphoid (CD19+), and T lymphoid cells (CD4+and CD8+) were significantly higher after co-cultivation of the human cells on AFT024 than on human stroma, prior to transplantation. Interestingly, the length of time in culture did not significantly affect the engraftment of the myeloid and T lymphoid lineage progenitors, but the percentage of B lymphoid lineage engraftment decreased significantly between 2 and 3 weeks of co-cultivation on both types of stroma. Cells with a primitive phenotype (CD45+/CD34-/CD38- and CD45+/CD34-/lin-) and cells with the capacity to generate secondary human CFU after recovery from the bnx bone marrow were maintained at significantly higher levels during culture on AFT024 stroma than on human stroma. The current studies demonstrate that the AFT024 murine stromal cell line supports the ex vivo survival and maintenance of human hematopoietic progenitors that are capable of long-term multilineage reconstitution for 2-3 weeks ex vivo, to levels superior to those that can be obtained using human stromal cells.