Aim: While stem cell research is accelerating rapidly towards clinical application, many issues still need to be resolved. One aspect often overlooked is cell attachment, which is critical for regenerative medicine purposes, and also for maintaining stem cell identity during expansion. To determine the effects and efficacy of attachment proteins, human stem cell (induced pluripotent, hiPSC; embryonic, hESC; adipose-derived, hASC) behavior was evaluated on laminin, fibronectin, Matrigel, and poly-l-lysine (PLL). Method: Stem cells were cultured on attachment protein-coated surfaces and traditional plates as a control (poly-styrene, PS). Cell growth was measured via cell counts, differentiation was monitored via gene expression, and morphometry measures were taken. Results: After 5 days, hASCs exhibited highest proliferation on laminin, followed by fibronectin, Matrigel, the control PS, and lastly PLL. hiPSCs proliferated most on Matrigel, closely followed by laminin; fibronectin, PLL, and PS resulted in very low to no cell counts. Although hiPSC numbers were slightly higher on Matrigel, cell colonies were larger on laminin by day 7. hESCs exhibited highest cell counts on Matrigel by day 4, however by day 8 cell numbers on laminin exceeded those on Matrigel. Gene expression and morphometry for all stem cell types showed maintenance of stem cell identity. Conclusions: hASCs prefer laminin for cell attachment and proliferation. hiPSCs prefer Matrigel for proliferation and laminin for migrating. hESCs seem to prefer Matrigel for short-term, rapid proliferation and laminin for long-term growth. These data show the importance of cell attachment and the selection of the appropriate protein for the desired stem cell and application.