Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs.