ABSTRACT Platelet counts are tightly regulated in order to prevent thrombotic or hemorrhagic complications associated with thrombocytosis or thrombocytopenia, respectively. While strides have been made in our understanding of proplatelet formation (PPF), there is still limited knowledge regarding the mechanisms through which the bone marrow (BM) extracellular matrix (ECM) regulates platelet production. Indeed, the BM includes a rich ECM with potential to generate mechanical constraints. Yet, the impact of BM mechanics on megakaryocyte (MK) properties and platelet formation has been understudied. Here, we propose an integrative approach to investigate emerging concepts related to the role of MK mechanobiological receptors in controlling MK adhesion to the ECM and platelet production. Our ultimate goal is to understand how specific MK mechanosensors sense the BM matrix to affect the cellular cytoskeleton, MK properties and, importantly, platelet level. Building upon our novel findings, Aim 1 explores the new paradigm and hypothesis that distinct MK cation channels preferentially respond to different BM matrix proteins and inversely impact the MK cytoskeleton and platelet levels. Experiments will focus on the Piezo family of cation channel mechanosensors, as compared to the Transient Receptor Potential cation channel subfamily V member 4 mechanosensor. In recent studies, we found these mechanosensors to have distinct preferences for different matrix proteins and opposing effects on PPF. Investigations will be carried out using pharmacological approaches as well as newly generated knockout mice at baseline and in response to challenges, such as myelosuppression or thrombocytopenia. Encouraged by preliminary studies using human primary MKs, continued studies will confirm murine findings. Aim 2 delineates mechanisms mediating novel connections between MK mechanosensors, integrin receptors activation, cytoskeletal changes, and MK mechano-sensitive transcription factors. This proposal is significant as there is need to identify new and alternative thrombopoietic pathways and agents that modulate platelet counts. In addition to conceptual innovation, at the technical level we will analyze new mouse models we developed with deletion of specific mechanosensors in MKs, and will apply state-of-the-art imaging and measurements under flow to follow cellular processes. Proposed studies are expected to yield new insights on the role of selective ECM sensing by MKs in controlling the MK cytoskeleton, adhesion and platelet production, with significant potential to impact our ability to modulate platelet levels.