Chemotherapy-induced hematopoietic toxicity is a multifactorial challenge in the treatment of oncology patients. The resultant bone marrow suppression is a major dose-limiting side effect. In this study, we utilized 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, to investigate the mechanisms underlying bone marrow recovery following chemotherapy. A single injection of 5-FU did not alter mouse bone structure but caused acute damage to bone marrow cellularity and vasculature. Single-cell RNA-sequencing of bone marrow mesenchymal lineage cells revealed a substantial reduction in early mesenchymal progenitors and a marked expansion of marrow adipogenic lineage precursors (MALPs) five days post-treatment. Furthermore, 5-FU upregulated the expression of myofibroblast markers in MALPs, indicating a myofibroblast transformation. Using Adipoq-Cre to label MALPs in vivo, we observed that 5-FU transiently increases the number of MALPs in the bone marrow by promoting their proliferation. Immunostaining confirmed the elevated expression of myofibroblast markers in MALPs. By day 14 after 5-FU injection, bone marrow cellularity and vasculature were largely restored
however, the ablation of MALPs significantly impaired this recovery. Taken together, our study uncovers the critical role of MALPs in facilitating bone marrow repair following chemotherapy-induced injury and identifies them as a potential cellular target for treating chemotherapy-induced myelosuppression.