Dominant mutations in the T-box transcription factor gene TBX5 cause Holt-Oram syndrome (HOS), an inherited human disease characterized by upper limb malformations and congenital heart defects (CHDs) of variable severity. We hypothesize that minor alterations in the dosage of Tbx5 directly influences severity of CHDs. Using a mouse allelic series, we show a sensitive inverse correlation between Tbx5 dosage and abnormal cardiac morphogenesis and gene expression. The CHDs found in mice harbouring a hypomorphic allele of Tbx5 (Tbx5(lox/+) mice) are less pronounced than those found in Tbx5 haploinsufficient mice (Tbx5(del/+)), and homozygous hypomorphic (Tbx5(lox/lox)) embryos have noticeably more advanced cardiac development than Tbx5 null (Tbx5(del/del)) embryos. Examination of target gene expression across the allelic series uncovers very fine sensitivity across the range of Tbx5 dosages, in which some genes respond dramatically differently to only 15% differences in Tbx5 mRNA levels. This analysis was expanded to a genome-wide level, which uncovered a Tbx5 dosage-sensitive genetic program involving a network of cardiac transcription factors, developmentally important cell-cell signaling molecules, and ion channel proteins. These results indicate an exquisite sensitivity of the developing heart to Tbx5 dosage and provide significant insight into the transcriptional and cellular mechanisms that are disrupted in CHDs.