NOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. Our data revealed an upregulation of Nsun5 at the 2-cell stage of mouse preimplantation development, suggesting its significance in early embryonic development. Given m5C's important role in stabilizing rRNA and mRNA and the Hippo signaling pathway's critical function in lineage segregation during embryogenesis, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. To examine this hypothesis, we employed Nsun5-specific small interfering RNAs for targeted gene silencing in mouse preimplantation embryos. Nsun5 knockdown resulted in significant developmental impairments including reduced blastocyst formation, smaller size of blastocysts, and impaired hatching from the zona pellucida. Nsun5 knockdown also led to decreased cell numbers and increased apoptosis in embryos. We also observed diminished nuclear translocation of yes-associated protein 1 (YAP1) in Nsun5 knockdown embryos at the morula stage, indicating disrupted cell differentiation. This disruption was further evidenced by an altered ratio of CDX2-positive to OCT4-positive cells. Furthermore, Nsun5 depletion was found to upregulate the Hippo signaling-related key genes, Lats1 and Lats2 at the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway.