Objective: To investigate the role of pseudouridine synthase 7 (PUS7) in neural lineage differentiation of mouse embryonic stem cells (mESCs) and to elucidate the involvement of mRNA pseudouridylation in this process. Methods: A Pus7-knockout mESC model was generated using CRISPR/Cas9. qRT-PCR, Western blotting, immunofluorescence, CCK-8 assays, and flow cytometric cell-cycle analysis were performed to evaluate the effects of Pus7 deficiency on pluripotency, proliferative capacity, and cell-cycle distribution in mESCs. Embryoid body (EB) differentiation and directed neural ectoderm differentiation were conducted to systematically assess the impact of Pus7 deficiency on lineage differentiation propensity. Differential bisulfite-induced deletion sequencing(BID-seq)and RNA sequencing(RNA-seq)analyses, together with pathway enrichment analysis, were further performed to identify potential mRNA pseudouridylation-regulated target genes. Results: Pus7 knockout did not markedly disrupt the core pluripotency network of mESCs but led to reduced cell proliferative capacity accompanied by G1-phase prolongation. During spontaneous EB differentiation, Pus7-deficient cells exhibited enhanced ectoderm-related programs, particularly neuroectodermal programs. During directed neural ectoderm differentiation, Pus7 deficiency enhanced neural progenitor cell programs, whereas maturation-related programs of neurons were impaired. Integrated BID-seq and RNA-seq analyses revealed that a subset of neural connectivity-related transcripts, such as neurexin 3(Nrxn3), ADAM metallopeptidase domain 23(Adam23), plexin D1(Plxnd1), solute carrier family 1 member 1(Slc1a1), unc-51 like autophagy activating kinase 4(Ulk4), and Abelson helper integration site 1 (Ahi1), displayed concurrent reductions in pseudouridylation and mRNA expression. Conclusion: Pus7 deficiency prolongs the G1 phase, enhances the neuroectodermal differentiation propensity of mESCs, and reduces the pseudouridylation and expression of neural connectivity-related mRNAs, thereby promoting neural progenitor cell programs while impairing subsequent maturation and disrupting the balance of neural lineage differentiation during embryonic development.