Mesenchymal stem cells (MSCs) participate in regenerative osteogenesis by generating bone-forming cells. To examine the proliferative capacity of MSC populations from bone marrow and their relationship to trauma severity (multiple trauma, monofracture, atrophic nonunion), we quantified colony properties of human MSCs in vitro. Serum levels of mediators associated with bone formation were also assessed. Fifty-five individuals were enrolled in this study (13 multiple trauma patients, 15 patients with monofracture, 20 patients with atrophic nonunions, 7 healthy volunteers). The colony forming unit-fibroblast (CFU-F) assay was used to quantify total colony number, mean cell density per colony, and mean colony area. MSC phenotype was established using flow cytometry and osteogenic differentiation. MSCs obtained from multiple-trauma patients yielded the highest reservoir. Significant differences in colony numbers of MSCs in female subjects were found between multiple-trauma patients (mean ± SD 48 ± 21 CFU-F/culture) and healthy volunteers (18.7 ± 3.3 CFU-F/culture, P < 0.05), patients with monotrauma (15 ± 10 CFU-F/culture, P < 0.05), and patients with atrophic nonunions (6.3 ± 4.1 CFU-F/culture, P < 0.05). In male participants, significant differences were found between patients with nonunions (14 ± 14 CFU-F/culture) and healthy volunteers (54 ± 17 CFU-F/culture, P < 0.05) as well as multiple-trauma patients (59 ± 25 CFU-F/culture, P < 0.05). The highest proliferative capacity (cell density) was seen in multiple-trauma patients. These data suggest that trauma severity and gender affect the reservoir and proliferation capacity of bone marrow-derived MSCs.