Over the past decade, membrane vesicles, known as exosomes, have garnered significant attention due to their potential as natural nanocarriers. Recently, it was discovered that plants also release extracellular vesicles (EVs). Yet, our understanding of EV production in plant cell culture remains limited. In this study, we used callus culture of the model plant Arabidopsis thaliana to isolate and comprehensively characterize EVs emanating from cultivated cells. Our findings reveal that the quantity of EVs in calli was significantly lower than in the plant's apoplastic fluid. This discrepancy is linked to the transcriptional repression of the endosomal sorting complex required for transport (ESCRT) genes. While salicylic acid elevated the expression of ESCRT components, it did not boost EV production. Remarkably, EVs from calli encompass proteins pivotal for cell wall biogenesis and defense mechanisms, as well as microRNAs consistent with those in intact plants. This indicates that plant cell cultures might be a viable source of EVs reflecting the properties of the parent plant species. Nevertheless, additional research is crucial to pinpoint the optimal conditions for efficient EV production.