Tendon injuries are increasingly prevalent around the world, accounting for more than 100 000 new clinical cases/year in the USA alone. Cell-based therapies have been proposed as a therapeutic strategy, with recent data advocating the use of tendon stem cells (TSCs) as a potential cell source with clinical relevance for tendon regeneration. However, their in vitro expansion is problematic, as they lose their multipotency and change their protein expression profile in culture. Herein, we ventured to assess the influence of insulin-like growth factor 1 (IGF-1), growth and differentiation factor-5 (GDF-5) and transforming growth factor-β1 (TGFβ1) supplementation in TSC culture. IGF-1 preserved multipotency for up to 28 days. Upregulation of decorin and scleraxis expression was observed as compared to freshly isolated cells. GDF-5 treated cells exhibited reduced differentiation along adipogenic and chondrogenic pathways after 28 days, and decorin, scleraxis and collagen type I expression was increased. After 28 days, TGFβ1 supplementation led to increased scleraxis, osteonectin and collagen type II expression. The varied responses to each growth factor may reflect their role in tendon repair, suggesting that: GDF-5 promotes the transition of tendon stem cells towards tenocytes; TGFβ1 induces differentiation along several pathways, including a phenotype indicative of fibrocartilage or calcified tendon, common problems in tendon healing; and IGF-1 promotes proliferation and maintenance of TSC phenotypes, thereby creating a population sufficient to have a beneficial effect. Copyright © 2014 John Wiley & Sons, Ltd.