Abstract Background Generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) in vitro takes about 21 days, making it unaffordable for clinical applications. Acceleration of the in vitro erythropoiesis process by using small molecules could eventually make the large-scale production of these cells commercially viable. Transforming Growth Factor β1 (TGF-β1) has been shown to have a dose-dependent activity on the HSCs: at high concentration it inhibits, whereas at low concentration it stimulates the HSCs growth. At high concentration, it also inhibits erythropoiesis but accelerates terminal erythroid differentiation of cell lines and erythroid progenitors. Here we examined whether the use of low concentration of TGF-β1 would be beneficial for increasing RBC production by stimulating HSC growth and also supporting erythroid differentiation. Such a strategy could make RBC production in vitro more efficient and cost-effective for clinical applications. Methods HSCs isolated from Apheresis samples were differentiated into mature RBCs by the sequential addition of specific combinations of growth factors for 21 days. In the control set, only EPO (3 IU/ml) was added whereas, in the test set, TGF-β1 at a concentration of 10 pg/ml was added along with EPO (3 IU/ml) from day 0. Results We found that a low concentration of TGF-β1 has no inhibitory effect on the proliferation of the early stages of erythropoiesis. Additionally, it significantly accelerates terminal stages of erythroid differentiation by promoting BNIP3L/NIX-mediated mitophagy. Conclusions Incorporation of TGF-β1 at 10 pg/ml concentration in the differentiation medium accelerates the in vitro erythropoiesis process by 3 days. This finding could have potential applications in transfusion medicine.