The feeder free culturing of pluripotent stem cells aims to eliminate the use of mouse embryonic fibroblasts (MEFs) as substrate during in vitro culturing. Here, the potential of fibronectin and vitronectin to keep stem cells undifferentiated was compared using two different stem cell lines (HUES1 and HUES7). Cells were cultured on vitronectin and fibronectin for 3 weeks. Results showed that cells cultured in fibronectin appear to adhere slightly well initially while those in vitronectin detached and bulked up in culture. However, cells cultured on either substrate were well attached and expressed Oct4 and Nanog at the end of the 3-week protocols. This signals the capacity of both vitronectin and fibronectin to keep stem cells undifferentiated after a few passages. Such comparisons of stem cell culture substrates allow the maximization of stem cell culture conditions that are specific to the cell line in use.