In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl -/- mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl -/- p300 plt6/+ mice displayed elevated platelet counts relative to Mpl -/- p300 +/+ controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300 Plt6 mutation, or recipients of Mpl +/+ p300 Plt6/Plt6 bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl -/- thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte development, the inhibition of which allows substantial thrombopoietin (TPO)-independent platelet production.