We have previously isolated and identified stem cells from human cruciate ligaments. The goal of this study was to evaluate the proliferation and differentiation abilities of ligament-derived stem cells (LSCs) cultured with growth factors, including fibroblast growth factor 2 (FGF-2), epidermal growth factor, and transforming growth factor-beta 1 (TGF-b1). The ligament tissues were obtained from patients with anterior cruciate ligament injuries receiving arthroscopic surgeries. LSCs were obtained by collagenase digestion and plating as previously reported. Surface immunophenotype and the potential for trilineage differentiation into osteoblasts, chondrocytes, and adipocytes were confirmed. It was found that proliferation of the cells was enhanced with the addition of FGF-2 and TGF-b1. Upon TGF-b1 treatment, expression of collagen type I and type III, tenascin-c, fibronectin, and a-smooth muscle actin were significantly upregulated. Additionally, LSCs treated with TGF-b1 and FGF-2 increased the production of collagenous and noncollagenous extracellular matrix protein. Together, these results demonstrate that LSCs respond differently to various cytokines, and the results further validate the potential of using cruciate ligament tissue as a stem cell source for tissue engineering purposes.