Most cell therapy products require an ex vivo cell processing step in a polymeric culture device. The transition from open systems such as tissue culture polystyrene (TCPS) flasks to functionally closed systems such as cell culture bags often entails a change in materials in contact with cells. It is well established that cell-material interactions affect cell behavior and cell fate decisions. Here, we investigated the effect of different culture surface on monocyte-derived dendritic cells (Mo-DCs), a commercially relevant immunotherapy product. Monocytes were isolated from the peripheral blood of healthy donors and differentiated into Mo-DCs in fluoropolymer (FEP) bags and conventional TCPS vessels. Quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated rapid adsorption of albumin, insulin and transferrin proteins from the medium onto fluoropolymer surfaces to reach equilibrium within the first 2 minutes in the medium. Consistent with these observations, contact angle and atomic force microscopy experiments showed a significant increase in fluoropolymer surface hydrophilicity and a decrease in rigidity at the surface following incubation in cell culture medium. After 7 days of differentiation and 2 days of maturation, Mo-DCs obtained both in FEP and TCPS cell culture vessels expressed the surface markers for mature antigen-presenting cells based on flow cytometry. Overall, although FEP is much more hydrophobic than TCPS, differences in surface properties decrease after protein adsorption, leading to similar performance for the production of Mo-DCs.