Therapeutic resistance and infiltrative capacities justify the aggressiveness of glioblastoma. This is due to cellular heterogeneity, especially the presence of stemness-related cells, i.e. Cancer Stem Cells (CSC). Previous studies focused on autophagy and its role in CSCs maintenance; these studies gave conflicting results as they reported either sustaining or disruptive effects. In the present work, we silenced two autophagy related genes -either Beclin1 or ATG5- by shRNA and we explored the ensuing consequences on CSCs markers’ expression and functionalities. Our results showed that the down regulation of autophagy led to enhancement in expression of CSCs markers, while proliferation and clonogenicity were boosted. Temozolomide (TMZ) treatment failed to induce apoptotic death in shBeclin1-transfected cells, contrary to control. We optimized the cellular subset analysis with the use of Sedimentation Field Flow Fractionation, a biological event monitoring- and cell sorting-dedicated technique. Fractograms of both shBeclin1 and shATG5 cells exhibited a shift of elution peak as compared with control cells, showing cellular dispersion and intrinsic sub-fraction modifications. The classical stemness fraction (i.e. F3) highlighted data obtained with the overall cellular population, exhibiting enhancement of stemness markers and escape from dormancy. Our results contributed to illustrate CSCs polydispersity and to show how these cells develop capacity to bypass autophagy inhibition, thanks to their acute adaptability and plasticity.