A significant problem in flexor tendon repair is the lack of suitable graft material for reconstruction. The ex vivo production of flexor tendon graft constructs requires the expansion of primary cells. Growth factors, such as platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF), are known to promote tendon healing and tendon cell proliferation. The purpose of these experiments was to optimize tenocyte proliferation in 3 tendon cell populations using growth factor supplementation. Cells of the synovial sheath, epitenon, and endotenon were isolated from rabbit flexor digitorum profundus tendons and maintained in culture. Cell cultures were supplemented with IGF-1, PDGF-BB, and bFGF alone and in combination. The conditions used for individual growth factor supplementation were IGF-1 (10, 50, and 100 ng/mL), PDGF-BB (1, 10, and 50 ng/mL), and bFGF (0.5, 1, and 5 ng/mL). The conditions used for combinations of growth factors were IGF-1 + PDGF-BB (50 + 10 and 100 + 50 ng/mL, respectively) and IGF-1 + PDGF-BB+ bFGF (50 + 10 + 1; 50 + 10 + 5; 100 + 50 + 1; and 100 + 50 + 5 ng/mL, respectively). For all 3 tendon cell populations, proliferation at 72 h was greater in the presence of individual growth factors as compared to controls. With PDGF-BB (50 ng/mL) supplementation, mean absorbance values increased 97% (0.57 to 1.13) in S cells, 37% (0.51 to 0.70) in E cells, and 33% (0.33 to 0.44) in T cells ( p < 0.001). In addition, a synergistic effect was observed. The combination of growth factors resulted in greater proliferation as compared to maximal doses of individual growth factors. In cultures supplemented with IGF-1 (100 ng/mL) +PDGF-BB (50 ng/mL), mean absorbance increased 114% (0.57 to 1.22) in S cells, 63% (0.51 to 0.831) in E cells, and 47% (0.33 to 0.48) in T cells ( p < 0.001). IGF-1 (100 ng/mL) + PDGF-BB (50 ng/mL) + bFGF (5 ng/mL) resulted in the greatest amount of cell proliferation for all 3 tendon cell populations. The mean absorbances increased 251% in S cells, 98% in E cells, and 106% in T cells ( p < 0.001). In summary, IGF-1, PDGF-BB, and bFGF can be used in combination to maximize tenocyte proliferation. Synergism among growth factors may provide a means to facilitate tendon engineering.