Alkanethiol self-assembled monolayers (SAMs) have been used extensively in studying the role of surface functionality and geometry on stem cell differentiation; however, the effects of stem cell culture conditions on SAM stability over time are not well understood. In this work, we examined the physical and chemical changes occurring on gold (Au)-SAM surfaces over time as a function of Au thickness. Within a narrow range of thicknesses (4, 8, and 10 nm), we observed significant differences in temporal SAM stability for a commonly utilized, hydrophilic, protein and cell repulsive oligo(ethylene) glycol alkanethiol (HS-(CH2 )11 (O(CH2 )2 )6 OH) SAM and the hydrophobic, protein adhesive hexadecanethiol (SH-(CH2 )15 CH3 ) SAM. Within both acellular and stem cell culture conditions, 8 nm Au resulted in the most stable SAMs (∼7 days). The 4 and 10 nm Au SAMs exhibited loss in stability following 5 days at varying degradation rates, showing 4 nm Au to be the least stable. Migration of human mesenchymal stem cells seeded on SAM surfaces showed that SAM degradation rates in acellular conditions were directly correlated with the cellular migration behavior. Findings of this study can be used to develop SAM surfaces with controlled degradation rates for applications in stem cell engineering and regenerative medicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 464-474, 2017.