During early mammalian embryogenesis, totipotent zygotes and early blastomeres undergo extensive post‐transcriptional regulation during the establishment of the first cell lineages; however, the functional contribution of alternative splicing to embryonic compaction and blastulation remains poorly understood. Here, we show that SF3B1, a core component of the spliceosome, is upregulated from the 4‐cell stage and mediates highly dynamic splicing programs. Depletion of SF3B1 results in developmental arrest at the morula stage, accompanied by widespread transcriptomic dysregulation characterized by aberrant expression of transcription factors that impede pluripotency transition. Alternative splicing analysis further identifies that aberrantly spliced transcripts were significantly enriched in genes involved in cell cycle regulation, such as Cdk11b and Ccnb1 . Importantly, we demonstrate that SF3B1 undergoes intrinsic, IDR‐driven liquid‐liquid phase separation both in vitro and in vivo, forming nuclear condensates in oocytes and early embryos, which is essential for successful development to the blastocyst stage. Together, our findings reveal that phase separation mediated SF3B1 splicing activity is a critical regulator of early mouse embryonic development.

Liquid-liquid phase separation (LLPS) regulates protein function by organizing proteins into dynamic condensates, yet how divergence in LLPS among orthologs gives rise to species-specific functional outputs remains poorly understood. Here, we show that intrinsically disordered regions (IDRs) act as transferable modules whose sequence composition encodes phase separation capacity and functional outputs. Incorporating the QPGY domain from human SS18 into its yeast ortholog Snf11 yields a chimeric protein (Snf11-QPGY) that forms dynamic intracellular condensates. These condensates activate the adhesion genes FLO1 and FLO11, triggering calcium-dependent flocculation absent in wild-type cells. Mechanistically, Snf11-QPGY sequesters histone deacetylase subunit Rpd3 within condensates, reducing its occupancy at FLO promoters and promoting histone acetylation and transcriptional activation. Substituting QPGY with a heterologous LLPS-competent IDR restores condensate formation but does not recapitulate QPGY-driven transcriptional-phenotypic outputs. These findings reveal how evolutionary diversification of IDR sequences drives functional divergence among orthologs and offer a framework for engineering IDR-dependent regulatory modules.