Tumor-repopulating cells (TRCs) are a tumorigenic sub-population of cancer cells that drives tumorigenesis. We have recently reported that soft fibrin matrices maintain TRC growth by promoting histone 3 lysine 9 (H3K9) demethylation and Sox2 expression and that Cdc42 expression influences H3K9 methylation. However, the underlying mechanisms of how soft matrices induce H3K9 demethylation remain elusive. Here we find that TRCs exhibit lower focal adhesion kinase (FAK) and H3K9 methylation levels in soft fibrin matrices than control melanoma cells on 2D rigid substrates. Silencing FAK in control melanoma cells decreases H3K9 methylation, whereas overexpressing FAK in tumor-repopulating cells enhances H3K9 methylation. Overexpressing Cdc42 or RhoA in the presence of FAK knockdown restores H3K9 methylation levels. Importantly, silencing FAK, Cdc42, or RhoA promotes Sox2 expression and proliferation of control melanoma cells in stiff fibrin matrices, whereas overexpressing each gene suppresses Sox2 expression and reduces growth of TRCs in soft but not in stiff fibrin matrices. Our findings suggest that low FAK mediated by soft fibrin matrices downregulates H3K9 methylation through reduction of Cdc42 and RhoA and promotes TRC growth. (C) 2016 Elsevier Inc. All rights reserved.