A large volume culture system was developed for the ex vivo expansion of CD34 positive (+) hematopoietic progenitors, using cell donated by 15 patients receiving high-dose chemotherapy with autologous hematopoietic progenitor cell support (AHPCS). Substantial expansion of myeloid (181-fold) and megakaryocyte (41-fold) progenitors cells was demonstrated, using the conditions that we determined to be optimal: CD34+ progenitors cultured unperturbed for 7 (marrow) or 10 (blood) days in Teflon-coated bags with X-Vivo-10 medium containing 10% autologous plasma, 100 ng/ml, respectively, of recombinant stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The studies demonstrated that (a) CD34 selection was necessary to obtain large, clinically relevant numbers of hematopoietic progenitors, (b) the addition of G-CSF to the baseline regimen of SCF/IL-3/IL-6 significantly enhanced the expansion of myeloid progenitors, (c) the addition of IL-1 to SCF/IL-3/IL-6 did not significantly enhance myeloid progenitor cell expansion, (d) CD34+ G-CSF-mobilized peripheral blood progenitor cells (PBPC) produced higher numbers of myeloid progenitors in culture than CD34+ marrow cells, and (e) long-term tissue culture (LTC) assays demonstrate the preservation of long-term initiating cells in ex vivo culture. The short-term and long-term reconstituting capability of CD34+ PBPC cultured in this system remains to be determined and will be evaluated in a clinical trial where they will be used as the sole source of AHPCS following high-dose therapy.