Whether epidermal stem cells (SCs) maintain their phenotype in vitro remains largely unknown. To determine whether adult human SCs maintain their self-renewal behavior in vitro, we observed adult keratinocytes in vitro. We studied live cell imaging of freshly obtained normal adult human keratinocytes from surgical discards over 11-16 days. We then tracked each colony and defined cells that divided as P (proliferating) and cells that did not divide (in 48h) as D (differentiated). The three outcomes of divisions were classified as PP, PD, and DD. We were able to define SCs, as was done for neonatal keratinocytes (Roshan et al, 2016), by 100% PP divisions over the first 3 generations. SCs formed significantly larger colonies, as expected. Committed progenitor colonies mostly terminally differentiated (75%) during the observation period, whereas no SC colonies did. Over the period of observation, we found that in SC colonies PP divisions dominated (82.8%±6.5), even at 10-14 generations, while in committed progenitor colonies PP divisions were only 30.6%±4.0 of divisions (p<0.0001). Our relatively long observation (up to 16 days) allowed us to determine that SCs maintained a high proportion (>70%) of PP divisions over time. In conclusion, adult human epidermal SCs preserved their self-renewal behavior in culture. Further studies are needed to determine further properties of keratinocyte SCs in vitro.