The effects of different cytokines on growth of human cord blood CD34 cells was studied by performing long-term culture (LTC) with primary human stromal cells transduced with genes for either Flt3-ligand (L) (human transmembrane, murine soluble or murine membrane-bound forms), human interleukin 3 (IL-3) or human GM-CSF. Molecular analysis of genomic DNA from transduced stromal cells using neo-specific polymerase chain reaction demonstrated gene transfer of G418-selected stromal cell populations. Enzyme-linked immunosorbent assay and biological assays of conditioned media from transduced stromal cells indicated expression and release of soluble cytokines. Numbers of both immature and more mature progenitors (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GEMM, BFU-E, CFU-GM) were increased threefold compared to control in the Flt3-L (transmembrane) LTC throughout five weeks of culture. IL-3 and GM-CSF feeders increased progenitor cell output also, but these effects were significantly lower than Flt3-L feeders. The two Flt3-L isoform engineered feeders, Ex6 (soluble isoform) and 5H (membrane-bound isoform), showed a decreased effect compared to the transmembrane Flt3-L feeders and, in particular, Ex6 feeders were similar to control feeders and 5H feeders were comparable to Flt3-L feeders only in the first two weeks of LTC. These results were apparent also by limiting dilution assays that showed a higher frequency of pre-CFU in the transmembrane Flt3-L feeders compared to control and the other cytokine feeders. Exogenous addition of soluble growth factors to suspension cultures without feeder layers, while superior to stromal feeders for short-term expansion of early progenitors, were inferior to the long-term maintenance/output on stromal feeders. Pre-CFU analysis supported these data. These results may be of some significance to understanding the actions of Flt3-L on blood cell production.