There is heritability to interindividual variation in platelet count, and better understanding of the regulating genetic factors may provide insights for thrombopoiesis. MicroRNAs (miRs) regulate gene expression in health and disease, and megakaryocytes (MKs) deficient in miRs have lower platelet counts, but information about the role of miRs in normal human MK and platelet production is limited. Using genome-wide miR profiling, we observed strong correlations among human bone marrow MKs, platelets, and differentiating cord blood-derived MK cultures, and identified MK miR-125a-5p as associated with human platelet number but not leukocyte or hemoglobin levels. Overexpression and knockdown studies showed that miR-125a-5p positively regulated human MK proplatelet (PP) formation in vitro. Inhibition of miR-125a-5p in vivo lowered murine platelet counts. Analyses of MK and platelet transcriptomes identified LCP1 as a miR-125a-5p target. LCP1 encodes the actin-bundling protein, L-plastin, not previously studied in MKs. We show that miR-125a-5p directly targets and reduces expression of MK L-plastin. Overexpression and knockdown studies show that L-plastin promotes MK progenitor migration, but negatively correlates with human platelet count and inhibits MK PP formation (PPF). This work provides the first evidence for the actin-bundling protein, L-plastin, as a regulator of human MK PPF via inhibition of the late-stage MK invagination system, podosome and PPF, and PP branching. We also provide resources of primary and differentiating MK transcriptomes and miRs associated with platelet counts. miR-125a-5p and L-plastin may be relevant targets for increasing in vitro platelet manufacturing and for managing quantitative platelet disorders.