Recent studies evaluating hematopoietic stem cell (HSC) potential raise the possibility that, in addition to embryonic sources, adult valve fibroblasts may be derived from HSCs. To test this hypothesis, we used methods that allow the potential of a single HSC to be evaluated in vivo. This was achieved by isolation and clonal expansion of single lineage-negative (Lin⁻), c-kit⁺, Sca-1⁺, CD34⁻ cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) combined with transplantation of individual clonal populations derived from these candidate HSCs into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP⁺ cells within host valves. A subpopulation of these cells exhibited synthetic properties characteristic of fibroblasts, as evidenced by their expression of mRNA for procollagen 1α1. Further, we show by Y-chromosome–specific fluorescence in situ hybridization analysis of female-to-male transplanted mice that the EGFP⁺ valve cells are the result of HSC-derived cell differentiation and not the fusion of EGFP⁺ donor cells with host somatic cells. Together, these findings demonstrate HSC contribution to the adult valve fibroblast population.