Focal adhesion kinase is an important survival signal in cancer. Recently, we demonstrated that the autophosphorylation inhibitor of FAK, Y15, effectively inhibited cancer cell growth. We detected many cancer cell lines sensitive to Y15 and also detected several cell lines such as colon cancer Lovo-1 and thyroid K1 more resistant to Y15. We sought to determine the main players responsible for the resistance. To reveal the signaling pathways responsible for the increased resistance of these cancer cells to the inhibitor of FAK, we performed a microarray gene profile study in both sensitive and resistant cells treated with Y15 inhibitor to compare with the more sensitive cells. Among unique genes up-regulated by Y15 in Lovo-1 and K1 resistant cells, a stem cell marker-ALDH1A3-was detected to be up-regulated >twofold. The resistant Lovo-1 and thyroid K1 cells overexpressed ALDH1A3 and CD44 versus sensitive cells. Treatment with ALDH1A3 siRNAs or ALDH inhibitor, DEAB sensitized resistant Lovo-1 and K1 cells to Y15 inhibitor, decreased viability and caused G1 cell cycle arrest more effectively than each agent alone. In addition, down-regulation of CD44 that was overexpressed in resistant Lovo-1 cells with CD44 siRNA effectively decreased the viability of cells in combination with Y15. In addition, down-regulation of overexpressed MDR1 with specific inhibitor, PSC-833, also sensitized resistant colon cancer cells to Y15. This report clearly demonstrates the mechanism of resistance to FAK autophosphorylation inhibitor and the mechanism to overcome it that is important for developing FAK-targeted therapy approaches.