〔Abstract〕 Objective To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further investgation of the specific role of Spi1-encoded protein PU.1. Methods The Lck-Cre mice were mated with Spi1flox/floxmice to obtain Lck-Cre×Spi1flox/floxmice(T lymphocyte-specific Spi1 knockout mice),and the genotype was determined by polymerase chain reaction(PCR) and agarose gel electrophoresis. Magnetic beads were used to sort out the splenic T lymphocytes, and the knockdown efficiency of PU.1 in T cells was detected by Western blot, quantitative real-time PCR(qPCR) and flow cytometry. Results The Lck-Cre×Spi1flox/floxmouse genotype was stably inherited. Compared with Spi1flox/floxmice, the expression level of PU.1 was significantly reduced in splenic T cells of Lck-Cre×Spi1flox/floxmice. Conclusion In this study, the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology, which provided a reliable animal model for the subsequent experiments of the specific role of PU.1 in T cell-related diseases.