Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis, and cancer recurrence. CSCs are considered derived from normal stem cells affected by the inflammatory microenvironment. Stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from stem cells in the cancer-inducing niche, which is a condition of chronic inflammation rich in growth factors, interleukins, chemokines, etc. Exosomes are considered to be the key mediators responsible for the cell-to-cell communications carrying proteins, nucleic acids, metabolites, etc., to shuttle between cells. If these cells are in the environment of chronic inflammation, the exosomes should be reflecting the conditions. In this chapter, we detail the method of CSC initiation using extracellular vesicles (EVs) derived from cancer cell. The stem cells treated with the EVs acquired characteristics of CSCs showing spheroids expressing stemness markers in the suspension culture and high tumorigenicity in Balb/c nude mice. EVs might perform as suitable inducer for initiating CSCs from stem cells or progenitor cells. The model of CSCs and the procedure of their establishment with EVs will help study the exact effect of EVs in the cancer-inducing niche and tumor microenvironment.