The T-box transcription factor Tbx5 can interact with Nkx2.5 and Gata4 transcription factors to synergistically regulate heart-specific genes in the nucleus. While a nuclear role for Tbx5 is clearly defined, we have previously shown that Tbx5 shuttles from nuclear to cytoplasmic sites, forming a complex with the PDZ-LIM protein LMP4 on the actin cytoskeleton. In this study, using a developmental series of chicken hearts, we provide the first evidence for differential Tbx5 protein expression and sub-cellular localization during cardiogenesis. At the tissue level, we show temporally and spatially restricted Tbx5 co-expression with LMP4. In cells co-expressing LMP4 and Tbx5 we demonstrate dynamic Tbx5 re-localization from exclusively nuclear to nuclear and cytoplasmic expression in the atrio-ventricular cushion. Furthermore, in coronary vessel development we show exclusive cytoplasmic localization of Tbx5, indicating a function for Tbx5 in the cytoplasm. In addition, we discover unknown regulation of Tbx5 and LMP4 expression in epicardial tissue, suggesting a specific role for Tbx5 in epicardial formation. These studies provide in vivo significance of the LMP4/Tbx5 protein interaction, suggesting both nuclear and cytoplasmic roles for Tbx5. The shuttling between nuclear and cytoplasmic sites reveals a novel mechanism for Tbx transcription factor regulation in chicken heart development allowing new insights for a better understanding of the molecular basis of hand/heart birth defects associated with TBX5 mutations.