Thymosin β4(Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells(m ESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on m ESCs. Target genes during m ESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the m ESCs-derived cardiomyocytes. It was found that Tβ4 decreased m ESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, m ESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these m ESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant ?-catenin. Under m ESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of m ESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed m ESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation. However, its role in murine embryonic stem cells (m ESCs) proliferation and cardiovascular differentiation remains unclear. Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio- Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the m ESCs-derived cardiomyocytes. It was found that Tβ4 decreased m ESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, m ESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of Self-renewal ability in these m ESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant differences found in BMP2 / BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant? -catenin. Under m ESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α -SMA. Neither the electrophysiological properties of m ESCs-derived cardiomyocytes nor the hormonal regulation by Iso / Cch was affected by Tβ4. In conclusion, Tβ4 suppressed m ESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.