Human embryonic stem cell (hESC) growth is dependent on various factors released by feeder cells. Some of them have already been elucidated, although much research is still needed to understand the biology of stem cell maintenance in culture. Traditionally, primary mouse embryonic fibroblasts (PMEFs) have been used as feeder layers, and both murine and human fibroblast cell lines have been shown to support pluripotency and self-renewal of hESC. Here we report the derivation of three new mouse embryonic fibroblast cell lines, MEFLU-M, MEFLU-T, and MEFLU-TB, with different properties regarding growth and support for undifferentiated hESCs. MEFLU-TB is able to support continuous growth of the newly derived Man-1, as well as H1, HUES-1, HUES-7, HUES-8, and HUES-9 human embryonic stem cell lines. After more than 50 passages and doublings, MEFLU-TB feeders compare to early passage primary mouse embryonic fibroblasts in their ability to support undifferentiated hESC growth. Our results contradict a previous paradigm that PMEFs tend to lose their capacity to support proliferation of hESCs with increasing passages, and show that the MEFLU-TB mouse embryonic fibroblast cell line and its conditioned medium have the potential to support the maintenance of hESC lines. Also, our results clearly show that spontaneous immortalization of primary fibroblasts can be achieved in culture without any chemical addition or genetic modification.