〔Abstract〕 Objective To construct B cell conditional knockout Spi1 gene mice and analysis their genotypes to provide an animal model basis for disease pathogenesis and drug target research. Methods Mb1-cre transgenic mice were crossbred with Spi1flox/flox mice,. The genotypes of the offspring mice were identified using polymerase chain reaction (PCR) and agarose gel electrophoresis. The mice with the genotype of Mb1-cre×Spi1flox/flox that were screened out will be the homozygous mice with B cell-specific conditional knockout of the Spi1 gene. Results PCR identification results showed that when using flox primers for detection, only a 220 bp band was amplified, indicating a genotype of Spi1flox/flox in the mice. When using Mb1-Cre primers for detection, a 383 bp band was amplified, indicating a genotype of Mb1-cre×Spi1flox/flox in the mice; Flow cytometry results showed that compared to Spi1flox/flox mice, Mb1-cre×Spi1flox/flox mice exhibited significantly reduced PU.1 expression levels in bone marrow-derived mononuclear cells, peripheral blood mononuclear cells, and spleen-derived B cells. Moreover, PU.1 exhibited normal expression in other immune cells, such as CD4+ T cells and macrophages. Conclusion By utilizing the Cre/LoxP system and CRISPR/Cas9 technology, this study successfully generates mice with B cell-specific conditional knockout of the Spi1 gene, providing a reliable animal model for in-depth exploration of the specific role of PU.1 in B cell-related diseases.