The serum response transcription factor (Srf) controls developmental programs and proliferation, differentiation, cellular shape and cytoskeleton organization. Its activity is regulated through the mitogen-activated protein kinase pathway (MAPK)/Erk pathway and the GTPases Rho/mDia/actin pathway, allowing integration of extracellular mitogenic signals with changes in actin cytoskeleton. The Srf co-activators myocardinrelated transcription factors (Mrtfs) assist Srf in the expression of genes linked to actin cytoskeleton dynamics. Among Mrtfs, the megakaryocytic acute leukemia (MAL)(also known as Mrtf-A and megakaryoblastic leukemia-1) is an actin-binding protein whose nuclear accumulation is controlled by the actin treadmilling cycle. 1, 2 The human MAL gene is fused to the OTT/RBM15 gene in acute megakaryoblastic leukemia (AMKL) 3, 4 and a contribution of Mal activity to the late steps of murine and human megakaryopoiesis has been reported. 5, 6 Although the lack of Srf results in major cellular defects and embryonic lethality (reviewed in Miano et al. 7), Mal germ line deficiency results in a limited myoepithelial cells phenotype8, 9 probably because of the redundancies of Mrtf proteins. To gain insight into the role of Srf and Mal in megakaryocyte (Mk) development, we compared the defects induced by their lossof-function in adult hematopoietic cells. Srf flx mice10 were bred with polyinosine-polycytidine-inducible Mx1-Cre mice, 11 then analyzed 5 weeks post-induction (Supplementary Figure 1). The Mal/Mrft-AÀ/À mice have been reported. 9 Null animals showed thrombocytopenia with 72 and 25% of wild-type average platelet counts for MalÀ/À and SrfD/D; MxÀCre respectively (Figure 1a). In vivo, Mal-and Srf-null platelet half-life was similar to control, indicating that the thrombocytopenia was not the result of an accelerated clearance (Supplementary Figure 2). In spite of the thrombocytopenia, immunochemical (Figure 1b) and flow cytometric (Figure 1c) analyses showed accumulation of bone marrow (BM) Mks in null animals. Srf and Mal were dispensable for the control of ploidy (Supplementary Figure 3). However, both mutant cells showed a diminished capacity to form proplatelet (PP) structures in culture medium, with less than half of them showing short cytoplasmic extensions resembling PPs, when compared with normal counterparts (Figure 1d). Transmission electron microscopy revealed major alterations in Srf-null Mks morphology with cytoplasmic fragments scattered throughout the BM space and naked nuclei, suggesting that Mks had fragmented in situ (Figure 2iv). No platelets were observed, further underscoring an abnormal fragmentation within the marrow. Circulating platelets had an altered morphology with an increase in mean platelet volume of around 30%(Figure 1a) and the macrothrombocytopenia was confirmed by ultra-structure (Figure 2viii). Morphology of Mal-null Mks was less altered. A majority of them were extremely mature, with well-developed demarcation membranes and randomly scattered-granules, and showed some cytoplasmic extensions with a thin outer ring of cytoplasm suggesting that they were beginning to form PPs in the marrow; very few showed a smooth surface (Figure 2ii). In agreement, numerous deposits of von Willebrand factor (vWF) immunostaining were scattered in BM space, indicating that local platelet production had occurred (Figure 1b). MalÀ/À platelets were associated with an elongated shape and less-granules than their wild-type counterparts (Figure 2vi). Flow cytometry analyses showed normal numbers and frequencies of megakaryocyte-erythroid progenitors (MEP; Supplementary …