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Stem cells have been widely used in tendon tissue engineering. The lack of refined and controlled differentiation strategy hampers the tendon repair and regeneration. This study aimed to find new effective differentiation factors for stepwise tenogenic differentiation. By microarray screening, the transcript factor  Fos  was found to be expressed in significantly higher amounts in postnatal Achilles tendon tissue derived from 1 day as compared with 7‐days‐old rats. It was further confirmed that expression of  Fos  decreased with time in postnatal rat Achilles tendon, which was accompanied with the decreased expression of multiply tendon markers. The expression of  Fos  also declined during regular in vitro cell culture, which corresponded to the loss of tendon phenotype. In a cell‐sheet and a three‐dimensional cell culture model, the expression of  Fos  was upregulated as compared with in regular cell culture, together with the recovery of tendon phenotype. In addition, significant higher expression of tendon markers was found in  Fos ‐overexpressed tendon stem/progenitor cells (TSPCs), and  Fos  knock‐down gave opposite results. In situ rat tendon repair experiments found more normal tendon‐like tissue formed and higher tendon markers expression at 4 weeks postimplantation of  Fos ‐overexpressed TSPCs derived nonscaffold engineering tendon (cell‐sheet), as compared with the control group. This study identifies  Fos  as a new marker and functional driver in the early stage teno‐lineage differentiation of tendon, which paves the way for effective stepwise tendon differentiation and future tendon regeneration. S tem  C ells  T ranslational  M edicine   2017;6:2009–2019

Fos Promotes Early Stage Teno‐Lineage Differentiation of Tendon Stem/Progenitor Cells in Tendon