Obesity and excess adiposity account for approximately 20% of all cancer cases; however, biomarkers of risk remain to be elucidated. While fibroblast growth factor-2 (FGF2) is emerging as an attractive candidate biomarker for visceral adipose tissue mass, the role of circulating FGF2 in malignant transformation remains unknown. Moreover, functional assays for biomarker discovery are limited. We sought to determine if human serum could stimulate the 3D growth of a non-tumorigenic cell line. This type of anchorage-independent 3D growth in soft agar is a surrogate marker for acquired tumorigenicity of cell lines. We found that human serum from cancer-free men and women has the potential to stimulate growth in soft agar of non-tumorigenic epithelial JB6 P+ cells. We examined circulating levels of FGF2 in humans in malignant transformation in vitro in a pilot study of n = 33 men and women. Serum FGF2 levels were not associated with colony formation in epithelial cells (r = 0.05, p = 0.80); however, a fibroblast growth factor receptor-1 (FGFR1) selective inhibitor significantly blocked serum-stimulated transformation, suggesting that FGF2 activation of FGFR1 may be necessary, but not sufficient for the transforming effects of human serum. This pilot study indicates that the FGF2/FGFR1 axis plays a role in JB6 P+ malignant transformation and describes an assay to determine critical serum factors that have the potential to promote tumorigenesis.