Objective An miR-100 knockout mouse model was established to preliminarily explore the effect of miR-100 gene deletion on the development of the mouse hematopoietic system. Methods By putting EIIa-Cre^- ; miR-100^{fl/ fl} mice and EIIa-Cre⁺ ;miR-100^{+/ +} mice were bred to produce EIIa-Cre⁺ ; miR-100^{fl/ fl} (miR-100^{-/ -} ) mice, that is, miR-100 knockout mice. The genotype of mice was identified by PCR and q-PCR, and the knock-out efficiency of miR-100 gene in mouse bone marrow and spleen was verified. Peripheral blood, bone marrow, and spleen were isolated, and single-cell suspensions were prepared. The effect of gene deletion on the hematopoietic system of mice was analyzed by blood count, flow cytometry, and methylcellulose colony formation experiments. The relationship between miR-100 and Mospd2 was verified by q-PCR and RNA-binding protein immunoprecipitation (RIP). Results The PCR and q-PCR result showed that miR-100-knockout mice were successfully constructed. The result of the blood count, flow cytometry, and methylcellulose blood cell colony formation experiments showed that the proportion of peripheral blood myeloid cells in miR-100 gene-deficient mice increased, but mouse bone marrow and spleen myeloid cells, T/ B lymphocyte, and the proportional and absolute cell counts of lymphocytes and erythrocytes were unchanged, and deleting miR-100 had no effect on the proportion or number of bone marrow hematopoietic stem progenitor cells or the ability of bone marrow mononuclear cells to form colonies. The q-PCR result showed that miR-100 deletion promoted the expression of Mospd2 in mouse bone marrow cells. RIP experiments showed that miR-100 binds to Mospd2 in the form of an AGO2 protein complex and thereby regulates the expression of Mospd2. Conclusions A miR-100-knockout mouse model was successfully established in this study, and the gene deletion affected the proportion of peripheral blood myeloid cells, which verified the relationship between miR-100 and Mospd2. This study has provided further information on the role of this gene in the regulation of hematopoiesis in mice.