Recent studies reveal that the mechanical environment influences the behavior and function of various types of cells, including stem cells. However, signaling pathways involved in the mechanical regulation of stem cell properties remain largely unknown. Using polyacrylamide gels with varying Young's moduli as substrates, we demonstrate that mouse embryonic stem cells (mESCs) are induced to differentiate on substrates with defined elasticity, involving the Src-ShcA-MAP kinase pathway. While the dual inhibition of mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), termed "2i," was reported to sustain the pluripotency of mESCs, we find it to be substrate elasticity dependent. In contrast, Src inhibition in addition to 2i allows mESCs to retain their pluripotency independent of substrate elasticity. The alter-native dual inhibition of Src and GSK3 ("alternative 2i") retains the pluripotency and self-renewal of mESCs in vitro and is instrumental in efficiently deriving mESCs from preimplantation mouse embryos. In addition, the transplantation of mESCs, maintained under the alternative 2i condition, to immunodeficient mice leads to the formation of teratomas that include differentiation into three germ layers. Furthermore, mESCs established with alternative 2i contributed to chimeric mice production and transmitted to the germline. These results reveal a role for Src-ShcA-MAP kinase signaling in the mechanical regulation of mESC properties and indicate that alternative 2i is a versatile tool for the maintenance of mESCs in serum-free conditions as well as for the derivation of mESCs. STEM CELLS 2012;30:1394-1404