We report a critical role for the T-box transcription factor Tbx5 in development and maturation of the cardiac conduction system. We find that Tbx5 is expressed throughout the central conduction system, including the atrioventricular bundle and bundle branch conduction system. Tbx5 haploinsufficiency in mice ( Tbx5 del/+ ), a model of human Holt–Oram syndrome, caused distinct morphological and functional defects in the atrioventricular and bundle branch conduction systems. In the atrioventricular canal, Tbx5 haploinsufficiency caused a maturation failure of conduction system morphology and function. Electrophysiologic testing of Tbx5 del/+ mice suggested a specific atrioventricular node maturation failure. In the ventricular conduction system , Tbx5 haploinsufficiency caused patterning defects of both the left and right ventricular bundle branches, including absence or severe abnormalities of the right bundle branch. Absence of the right bundle branch correlated with right-bundle-branch block by ECG. Deficiencies in the gap junction protein gene connexin 40 ( Cx40 ), a downstream target of Tbx5 , did not account for morphologic conduction system defects in Tbx5 del/+ mice. We conclude that Tbx5 is required for Cx40 -independent patterning of the cardiac conduction system, and suggest that the electrophysiologic defects in Holt–Oram syndrome reflect a developmental abnormality of the conduction system.