Megakaryocytes (MK) are bone marrow cells responsible for platelet production. During maturation, MK undergo DNA replication without cytokinesis to become large in size (>65µm) and polyploid (≤128 N) but the molecular mechanisms underlying MK maturation are poorly understood. Our work has revealed that MK of individual ploidies exist as two distinct populations distinguished by set of cell surface markers such as CD41 and CD61. When CD41/CD61bright MK that are larger in size and have a distinct complex/granular cytoplasm were transplanted into WT mice, they released 500x more platelets than CD41/CD61dim MK. Intriguingly, we have identified a key role for the RNA binding protein Serine-arginine Rich Splicing Factor 3 (SRSF3) in megakaryopoiesis. In Srsf3-Pf4-Cre mice, where SRSF3 expression is ablated in MK, the CD41/CD61bright MK population is largely missing, resulting in severe thrombocytopenia with a 90% reduction in platelets. This together with our ultrastructural analysis of Srsf3-null MK using transmission electron microscopy supports our hypothesis that CD41/CD61bright MK subpopulation is responsible for platelet production. RNA-sequencing analysis of 8N versus >16N Srsf3-null and WT MK demonstrated that RNA processing mediated by SRSF3 plays a central role in tuning the MK transcriptome. We identified a large shift in RNA repertoire during MK maturation that did not take place in Srsf3-null MK. Moreover, platelets derived from Srsf3-Pf4-Cre mice were morphologically and functionally abnormal and had 30 times more RNA compared to WT platelets. Platelet RNA-sequencing showed that the Srsf3-null platelets had greatly abnormal RNA composition, with hundreds of up- and downregulated RNAs. Our work sheds light into the role of RNA processing in megakaryopoiesis and platelet biogenesis which may help in identifying new approaches to tackle thrombogenic and haematopoietic disorders.