Atomic force microscopy (AFM) provides the opportunity to perform fundamental and mechanistic observations of complex, dynamic, and transient systems and ultimately link material microstructure and its evolution during tribological interactions. This investigation focuses on the evolution of a dynamic fluoropolymer tribofilm formed during sliding of polytetrafluoroethylene (PTFE) mixed with 5 wt% alpha-phase alumina particles against 304L stainless steel. Sliding was periodically interrupted for AFM topography scans. The average film roughness, the average friction coefficient, and polymer wear rate based on sample height recession were recorded as a function of increasing sliding cycles. Topographical maps suggested tribofilm nucleates in grooves of the steel countersample, spreads, and develops into a uniform film through sliding. Prominent nanoscale features were visible around 10,000 sliding cycles and thereafter. Scanning electron microscopy and energy-dispersive X-ray spectroscopy showed good correlations between these features and aluminum-rich domains, suggesting the presence of alumina particles on the surface.