Uterine fibroids are the most prevalent benign tumors in women of reproductive age. The current knowledge on the fibroid disease mechanism has derived from studies of the Eker rat model where a unique germ line defect in the tuberous sclerosis 2 (Tsc2) tumor suppressor gene leads to the development of leiomyosarcoma, leiomyoma, and renal cancer. To study fibroids of human origin, we sought to establish fibroid xenografts in immune-compromised mice. We determined that lentiviral-mediated transduction of a green fluorescence protein (GFP)-luciferase (LUC) fusion gene and bioluminescence-based whole animal imaging allowed for the monitoring of transplanted fibroid cells in mice. We used this in vivo imaging approach to test a series of transplantation protocols and found that only freshly dissociated fibroid cells, but not the fibroid-derived smooth muscle cells grown in ex vivo cultures, can generate stable xenografts in subcutaneous Matrigel implants. Formation of the fibroid-xenografts requires the implantation of 17βestradiol-releasing pellets in the recipient mice. Furthermore, freshly dissociated myometrial cells do not form xenografts under the experimental conditions. The xenograft protocol developed from this study provides an avenue for investigating the pathogenesis and drug responses of human fibroids.