AbstractTbx5 is a member of the T‐box family of transcription factors and is associated with Holt–Oram syndrome (HOS), a congenital disorder characterized by heart and limb defects. Although implicated in several processes during development, only a few genes regulated by Tbx5 have been reported. To identify candidate genes regulated by Tbx5 during heart development, a microarray approach was used. A cardiac‐derived mouse cell line (1H) was infected with adenoviruses expressing Tbx5 or β‐galactosidase and RNA was isolated for analysis using an Affymetrix gene chip representing over 39,000 transcripts. Real‐time reverse transcriptase‐polymerase chain reaction confirmed Tbx5 induction of a subset of the genes, includingnppa,photoreceptor cadherin,brain creatine kinase,hairy/enhancer‐of‐split related 2, andgelsolin. In situ hybridization analysis indicated overlapping expression of these genes withtbx5in the embryonic mouse heart. In addition, the effect of HOS‐associated mutations on the ability of Tbx5 to induce target gene expression was evaluated. Together, these data identify several genes induced by Tbx5 that are potentially important during cardiac development. These genes represent new candidate gene targets of Tbx5 that may be related to congenital heart malformations associated with HOS. Developmental Dynamics 235:2868–2880, 2006. © 2006 Wiley‐Liss, Inc.