Protracted thrombocytopenia and bleeding remain serious complications in bone marrow transplantation (BMT). Major progress has been made in facilitating myeloid and erythroid engraftment, but little has been made in accelerating thrombopoiesis post-BMT. We report that in vitro preincubation of T cell-depleted BM allografts with a combination of interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.1 microgram/mL each) (n = 8), for 3 days prior to infusion, expands megakaryocyte (MK) precursors. MK-progenitor proliferation was assessed in plasma clot colony assays and liquid cultures following pre-exposure to IL-3/GM-CSF. We observed a 2.8-fold increase in the number of colony-forming units-megakaryocyte (CFU-MK) (17.3 +/- 5.2 vs. 6.1 +/- 3.4) (p = 0.001) and a two-fold increase in burst-forming units-megakaryocyte (BFU-MK) (0.2 vs. 0.1) (p = 0.01) per 2 x 10(5) cells/mL compared to control BM samples cultured for 3 days in medium alone. In secondary cultures, the continued presence of IL-3 and GM-CSF increased the number of CFU-MK by 200-fold (p < 0.0001) over controls and by 9.7-fold over fresh BM. A 33-fold increase (p < 0.0001) in the number of BFU-MK was elicited compared to controls. In addition, IL-3 plus GM-CSF supported increased cellularity within the colonies. The presence of IL-3 or GM-CSF alone resulted in fewer MK colonies and fewer cells per colony than both cytokines combined. In liquid cultures, the percentage of cells expressing platelet glycoprotein (GP) IIb/IIIa in the continued presence of IL-3 and GM-CSF increased following preincubation, yielding a total of 16.0 +/- 2.3 x 10(4) MK/2 x 10(6) cells at day 10 of culture. We propose that ex vivo preincubation with IL-3 and GM-CSF can expand the number of MK precursors and may facilitate platelet recovery post-BMT.