Mesenchymal stem cells (MSCs) are effective in hematopoietic engraftment and tissue repair in stem cell transplantation. In addition, these cells control the process of hematopoiesis by secreting growth factors and cytokines. The aim of the present study is to investigate the effect of rat bone marrow (BM)-derived MSCs on the granulocyte differentiation of rat BM-resident C-kit+hematopoietic stem cells (HSCs). The mononuclear cells were collected from rat BM using density gradient centrifugation and MSCs and C-kit+HSCs were isolated. Then, cells were divided into two groups and differentiated into granulocytes; C-kit+HSCs alone (control group) and co-cultured C-kit+HSCs with MSCs (experimental group). Subsequently, the granulocyte-differentiated cells were collected and subjected to real-time PCR and Western blotting for the assessment of their telomere length (TL) and protein expressions, respectively. Afterwards, culture medium was collected to measure cytokine levels. CD34, CD16, CD11b, and CD18 granulocyte markers expression levels were significantly increased in the experimental group compared to the control group. A significant change was also observed in the protein expression of Wnt and β-catenin. In addition, MSCs caused an increase in the TL of granulocyte-differentiated cells. MSCs could affect the granulocyte differentiation of C-kit+HSCs via increasing TL and Wnt/β-catenin protein expression.