Toll-like receptor (TLR) activation is important in immune responses and in differentiation of hematopoietic stem cells. We detected mRNA expression of TLRs 1, 2, 3, 5, and 6, but not TLRs 4, 7, 8, and 9 in murine (m)ESC line E14, and noted high cell surface protein expression of TLR2, but not TLR4, for mESC lines R1, CGR8, and E14. ESC lines were cultured in the presence of leukemia inhibitory factor (LIF). Pam(3)Cys enhanced proliferation and survival of the 3 ESC lines. In contrast, lipopolysaccharide (LPS) decreased proliferation and survival. Pam(3)Cys and LPS effects on proliferation and survival were blocked by antibody to TLR2, suggesting that effects of both Pam(3)Cys and LPS on these mESC lines were likely mediated through TLR2. E14 ESC line expressed MyD88. Pam(3)Cys stimulation of E14 ESCs was associated with induced NF-kappaB translocation, enhanced phosphorylation of IKK-alpha/beta, and enhanced mRNA, but not protein, expression of tumor necrosis factor-alpha, interferon-gamma, and IL-6. TLR2 activation by Pam(3)Cys or inhibition by LPS was not associated with changes in morphology or expression of alkaline phosphatase, Oct4, SSEA1, KLF4, or Sox2, markers of undifferentiated mESCs. Our studies identify TLR2 as present and functional in E14, R1, and CGR8 mESC lines.