Rationale: Thymosin beta 4 (Tβ4) is a 43–amino acid factor encoded by an X-linked gene. Recent studies have suggested that Tβ4 is a key factor in cardiac development, growth, disease, epicardial integrity, and blood vessel formation. Cardiac-specific short hairpin (sh)RNA knockdown of tβ4 has been reported to result in embryonic lethality at E14.5–16.5, with severe cardiac and angiogenic defects. However, this shRNA tβ4 -knockdown model did not completely abrogate Tβ4 expression. To completely ablate Tβ4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac-specific knockouts are critical. Objective: We examined the role of Tβ4 in developing and adult heart through global and cardiac specific t β4-knockout mouse models. Methods and Results: Global t β 4 -knockout mice were born at mendelian ratios and exhibited normal heart and blood vessel formation. Furthermore, in adult global t β 4 -knockout mice, cardiac function, capillary density, expression of key cardiac fetal and angiogenic genes, epicardial marker expression, and extracellular matrix deposition were indistinguishable from that of controls. Tissue-specific t β 4 -deficient mice, generated by crossing t β 4 -floxed mice to Nkx2.5-Cre and α MHC-Cre , were also found to have no phenotype. Conclusions: We conclude that Tβ4 is dispensable for embryonic viability, heart development, coronary vessel development, and adult myocardial function.