Single-cell transcriptional profiling has rapidly advanced our understanding of the embryonic hematopoiesis; however, whether and what role RNA alternative splicing (AS) plays remains an enigma. This is important for understanding the mechanisms underlying splicing-associated hematopoietic diseases and for the derivation of therapeutic stem cells. Here, we used single-cell full-length transcriptome data to construct an isoform-based transcriptional atlas of the murine endothelial-to-hematopoietic stem cell (HSC) transition, which enables the identification of hemogenic signature isoforms and stage-specific AS events. We showed that the inclusion of these hemogenic-specific AS events was essential for hemogenic function in vitro. Expression data and knockout mouse studies highlighted the critical role of Srsf2: Early Srsf2 deficiency from endothelial cells affected the splicing pattern of several master hematopoietic regulators and significantly impaired HSC generation. These results redefine our understanding of the dynamic HSC developmental transcriptome and demonstrate that elaborately controlled RNA splicing governs cell fate in HSC formation.