Cancer stem-like cells (CSCs) can generate solid tumors through the properties of stem cells such as self-renewal and differentiation and they cause drug resistance, metastasis and recurrence. Therefore, establishing CSC lines is necessary to conduct various studies such as on the identification of CSC origin and specific targeted therapies. In this study, we stimulated NIH3T3 fibroblasts to exhibit the characteristics of CSCs using the whole protein lysates of B16F10 melanoma cells. As a result, we induced colony formation that displayed self-renewal and differentiation capacities through anchorage-independent culture and re-attached culture. Moreover, colonies showed drug resistance by being maintained in the G0/G1 state. Colonies expressed various CSC markers and displayed high-level drug efflux capacity. Additionally, colonies clearly demonstrated tumorigenic ability by forming a solid tumor in vivo. These results show that proteins of cancer cells could transform normal cells into CSCs by increasing expression of CSC markers. This study argues the tremendous importance of the extracellular microenvironmental effect on the generation of CSCs. It also provides a simple experimental method for deriving CSCs that could be based on the development of targeted therapy techniques.