Thrombopoietin (TPO) is the major cytokine regulator of steady state platelet production and is required for rapid responses to platelet loss. TPO binding to its specific cell surface receptor, the Mpl protein, controls megakaryocyte number by stimulating production of megakaryocyte progenitor cells. TPO is also an important regulator of hematopoietic stem cells (HSC), required for maintenance of quiescence and self-renewal. Circulating levels of TPO are regulated by internalization and degradation of TPO via Mpl receptors on platelets and megakaryocytes providing an effective feedback regulatory mechanism whereby the availability of TPO is appropriate to the need for cellular production. To explore the mechanisms controlling platelet production, we have conducted mutagenesis screens for mutations that rescue the thrombocytopenia of c-Mpl-deficient mice. Loss of function mutations in the genes encoding c-Myb or its partner protein p300 result in supraphysiological platelet production in Mpl-/- mice with affected mice exhibiting expanded cellular production and skewed commitment in favour of megakaryopoiesis. Genomics studies in these mice have provided insights into pathways linking TPO signalling to stem cell regulation and megakaryopoiesis. To specifically explore the role of Mpl expression on megakaryocytes and platelets, both for cellular production and in feedback control, we generated a conditional knockout Mpl allele that included the ability to detect transcriptional activity of the locus via a green fluorescent protein (GFP) reporter. However, specific inactivation of Mpl in megakaryocytes and platelets using a platelet factor 4 cre-recombinase (PF4cre) transgene, resulted in an unexpected and a dramatic megakaryocytosis and thrombocytosis. Thus, TPO action via Mpl expression on megakaryocytes and platelets is dispensable for high-level platelet production. However, negative feedback regulation of available TPO by Mpl expression on megakaryocytes and platelets is essential in preventing excess production of Mpl-expressing stem and progenitor cells, that when unchecked drives dramatic megakaryocytosis and myeloproliferation.