Cancer-associated fibroblasts (CAF) are key contributors to malignant progression, but their critical regulators remain largely unknown. In this study, we examined the role of Twist1, a central regulator of epithelial–mesenchymal transition in carcinoma cells, in the transdifferentiation of normal quiescent fibroblasts to CAF and we defined its upstream controls and downstream effectors. Primary human gastric fibroblast and CAF cultures were established from gastrectomy specimens and validated as nontumor cells by somatic mutation analyses. In these cultures, exposure to the proinflammatory cytokine IL6 commonly expressed in tumors was sufficient to induce Twist1 expression in normal fibroblasts and transdifferentiate them into CAFs via STAT3 phosphorylation. In xenograft models, tumor infiltration of Twist1-expressing CAFs was enhanced strongly by ectopic IL6 expression in gastric or breast cancer cells. We found that Twist1 expression was necessary and sufficient for CAF transdifferentiation. Enforced expression of Twist1 in normal fibroblasts was also sufficient to drive CAF marker expression and malignant character in gastric cancer cells both in vitro and in vivo. Conversely, silencing the expression of Twist1 in CAFs abrogated their tumor-promoting properties. Downstream of Twist1, we defined the chemokine CXCL12 as a transcriptional target. Clinically, CXCL12 and Twist1 expression were correlated in CAFs present in gastric tumor specimens. Finally, ectopic expression of Twist1 in normal fibroblasts suppressed premature senescence, whereas Twist1 attenuation accelerated senescence in CAFs. Our findings define Twist1 as a compelling target to deprogram the tumor-supporting features of the cancer microenvironment. Cancer Res; 75(1); 73–85. ©2014 AACR.