Myofibroblasts are key players in kidney fibrosis, but their origin and function remain unclear. This study used genetically engineered mice to track and ablate myofibroblasts, revealing that 50% arise from local resident fibroblasts through proliferation, while 35% come from bone marrow via differentiation, and 10% from endothelial-to-mesenchymal transition (EndMT), and 5% from epithelial-to-mesenchymal transition (EMT). Targeting TGFβ1 signaling in αSMA+ cells reduced myofibroblast accumulation by 56%, but fibrosis decreased only by 29%, suggesting other pathways also contribute. Pericytes, including NG2+ and PDGFRβ+ cells, do not contribute to fibrosis, as their depletion did not reduce fibrosis or myofibroblast numbers. Myofibroblasts derived from bone marrow do not require proliferation for recruitment. EMT and EndMT contribute to myofibroblast formation, with EMT accounting for about 5% and EndMT for 10%. These findings highlight the complex origins of myofibroblasts in kidney fibrosis and suggest that targeting diverse pathways is necessary for effective treatment. The study provides new insights into the role of bone marrow and TGFβ1 in myofibroblast formation and fibrosis, and emphasizes the importance of understanding myofibroblast origins for developing therapeutic strategies.Myofibroblasts are key players in kidney fibrosis, but their origin and function remain unclear. This study used genetically engineered mice to track and ablate myofibroblasts, revealing that 50% arise from local resident fibroblasts through proliferation, while 35% come from bone marrow via differentiation, and 10% from endothelial-to-mesenchymal transition (EndMT), and 5% from epithelial-to-mesenchymal transition (EMT). Targeting TGFβ1 signaling in αSMA+ cells reduced myofibroblast accumulation by 56%, but fibrosis decreased only by 29%, suggesting other pathways also contribute. Pericytes, including NG2+ and PDGFRβ+ cells, do not contribute to fibrosis, as their depletion did not reduce fibrosis or myofibroblast numbers. Myofibroblasts derived from bone marrow do not require proliferation for recruitment. EMT and EndMT contribute to myofibroblast formation, with EMT accounting for about 5% and EndMT for 10%. These findings highlight the complex origins of myofibroblasts in kidney fibrosis and suggest that targeting diverse pathways is necessary for effective treatment. The study provides new insights into the role of bone marrow and TGFβ1 in myofibroblast formation and fibrosis, and emphasizes the importance of understanding myofibroblast origins for developing therapeutic strategies.