We report that blood cell autografts, collected by single leukapheresis in cancer patients (n = 11) at the time of mobilization of hematopoietic progenitors into peripheral blood following anticancer therapy with high-dose cyclophosphamide (HD-CTX) plus interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF/filgrastim), comprise 1.98 +/- 0.39 x 10(5)/kg (mean +/- SE) CD34+ progenitors of dendritic cells (DCs). This number corresponds to 140-fold more progenitors than in a control autograft collected in the steady state. DCs derived from mobilized CD34+ cells, morphologically and immunophenotypically undistinguishable from skin Langerhans cells and DCs from bone marrow and cord blood CD34+ cells, are shown to be powerful stimulators of allogeneic T cell proliferation in primary MLR and of autologous HLA-DR-restricted CD4+ T cell proliferation in response to presentation of xenogenic antigens. We show that the GM-CSF-plus-TNF-alpha-dependent ex vivo generation of DCs from mobilized CD34+ cells is 2.5-fold enhanced by flk-2/flt-3 ligand or c-kit ligand (stem cell factor) and five-fold enhanced by a combination of these growth factors. In addition, the optimal serum for the generation of DCs is autologous HD-CTX recovery-phase serum rather than fetal calf serum (FCS) or steady-state human serum, which are clinically inadequate and ineffective, respectively. In practice, the stimulation of CD34+ cells in a blood cell autograft (15.75 +/- 2.46 x 10(6)/kg) provided by the above four growth factors should permit ex vivo generation of approximately 40 x 10(9) DCs in an adult patient. These new findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.