Objective To breed and identify FOXO 3A gene knockout mice, hybrid mice were bred for conservation and homozygous mice were used for experiments. The effect of FOXO 3A gene knockout on the hematopoietic cell phenotype in mice was preliminarily analyzed, laying the foundation for subsequent studies on the regulation of the FOXO 3A gene during hematopoietic cell injury. Methods Hybrid female mice were mated with wild type male mice.Female and male hybrid mice were selected and bred, and homozygous mice were bred using one male and one or two females per cage. Pedicle marking and tail snips of neonatal mice were used to extract genomic DNA, and PCR was used for genotype identification. Heterozygous mice ( FOXO 3A+/ - ) with 100/186 bp bands, homozygous mice ( FOXO 3A-/ - ) with 186 bp bands, and wild type mice ( FOXO 3A+/ + , WT) with 100 bp bands were identified. Wild type and homozygous mice were used for bone marrow cell typing by flow cytometry. Results FOXO 3A gene knockout mice were successfully bred and identified. Homozygous and heterozygous mice from the daughters of gene knockout mice had normal growth, with no obvious difference in appearance or development compared with wild type FVB/ N mice. A number of homozygous mice were obtained for subsequent experiments. The bone marrow cell count of FOXO 3A knockout homozygous mice contained similar numbers of nucleated cells compared with wild type mice (6. 167±1. 424 vs 10±1. 732; P = 0. 1625). The results of flow analysis showed that the proportion of hematopoietic progenitor cells (Lin-Scal-1- c-kit+ , HPC) in the bone marrow of FOXO 3A knockout mice was significantly increased ( P <0. 05) compared with the wild type mice. In addition, there was no significant difference in the proportion of hematopoietic stem cells (Lin-Scal-1+ c-kit+ , HSC), or number of hematopoietic stem cells and hematopoietic progenitor cells ( P >0. 05). Conclusions FOXO 3A gene knockout mice were successfully obtained by genotype identification. Our preliminarily study demonstrated that FOXO 3A gene knockout had no significant effect on the bone marrow cell count or typing in mice. Future studies will investigate the effect of FOXO 3A gene knockout on hematopoietic cells in mice.