Transforming growth factor beta (TGF-beta) signals through activation of Smad transcription factors. Activated Smad proteins associate with different DNA-binding cofactors for the recognition and regulation of specific target genes. Members of the forkhead box O family (FoxO1, FoxO3, and FoxO4) play such a role in the induction of the cyclin-dependent kinase inhibitors p15Ink4b and p21Cip1. To delineate the organization of the TGF-beta response in human keratinocytes, we defined the set of genes whose activation by TGF-beta requires both FoxO and Smad functions. FoxO factors are shown to be essential for 11 of the 115 immediate gene activation responses to TGF-beta in these cells. FoxO1, FoxO3, and FoxO4 act redundantly as mediators of these effects. Smad4, which functions as a partner of receptor-phosphorylated Smad2/3, is required for all of these responses. These results define a FoxO-Smad synexpression group or group of genes that are jointly induced by a common mechanism in response to TGF-beta. In addition to p15INK4b and p21CIP1, these genes include mediators of stress responses (GADD45A, GADD45B, and IER1) and adaptive cell signaling responses (CTGF, JAG1, LEMD3, SGK, CDC42EP3, and OVOL1). Bioinformatic analysis of the promoter region of these genes reveals diverse configurations of Smad and FoxO binding elements, implying differences in the regulatory properties of this group of genes. Indeed, a subset of FoxO/Smad-dependent TGF-beta gene responses additionally require the transcription factor CCAAT/enhancer-binding protein beta. The composition of the FoxO-Smad synexpression group suggests that stress reactions and adaptive functions accompany the cytostatic response of keratinocytes to TGF-beta.