Hepatic stellate cells are believed to play a key role in the development of liver fibrosis. Several studies have reported that bone marrow cells can give rise to hepatic stellate cells. We hypothesized that hepatic stellate cells are derived from hematopoietic stem cells. To test this hypothesis, we generated chimeric mice by transplantation of clonal populations of cells derived from single enhanced green fluorescent protein (EGFP)–marked Lin⁻Sca-1⁺c-kit⁺CD34⁻ cells and examined the histology of liver tissues obtained from the chimeric mice with carbon tetrachloride (CCl₄)–induced injury. After 12 weeks of CCl₄ treatment, we detected EGFP⁺ cells in the liver, and some cells contained intracytoplasmic lipid droplets. Immunofluorescence analysis demonstrated that 50% to 60% of the EGFP⁺ cells were negative for CD45 and positive for vimentin, glial fibrillary acidic protein, ADAMTS13, and α-smooth muscle actin. Moreover, EGFP⁺ cells isolated from the liver synthesized collagen I in culture. These phenotypes were consistent with those of hepatic stellate cells. The hematopoietic stem cell–derived hepatic stellate cells seen in male-to-male transplants revealed only one Y chromosome. Our findings suggest that hematopoietic stem cells contribute to the generation of hepatic stellate cells after liver injury and that the process does not involve cell fusion.