The feeder layer constitutes a prerequisite for the undifferentiated proliferation of human embryonic stem (hES) cells in vitro. However, a few feeders have been reported to be non-supportive in nature, suggesting that these feeders exhibit a different transcriptome and proteome, in comparison to their supportive counterparts. In an attempt to identify factors required for undifferentiated growth and many downstream applications of hES cells, transcriptomes of supportive (mouse fibroblasts derived from 13.5dpc embryos and human fetal fibroblasts) and non-supportive (mouse fibroblasts derived from 18.5dpc embryos) feeders were analyzed. Furthermore, the parallel correlation of data generated in the microarray study with the published proteome data of supportive feeder fibroblasts, helped us to focus on the proteins which seem to be likely candidates in supporting the undifferentiated expansion of ES cells in vitro. Our results indicated that TGFbeta and its associated signaling molecules facilitate the undifferentiated proliferation of hES cells in vitro. The transient differentiation of feeder fibroblasts into myofibroblasts may be the decisive factor for a feeder layer to be supportive or non-supportive in nature. We propose that the microenvironment of feeder myofibroblasts dictates TGFbeta to support proliferation and apparently plays the contradictory role of facilitating differentiation when feeder support is withdrawn, possibly by acting through different signaling mechanisms.