Tumor microenvironment composition and architecture are known as a major factor in orchestrating the tumor growth and its response to various therapies. In this context, in vivo studies are necessary to evaluate the responses. However, while tumor cells can be of human origin, tumor microenvironment in the in vivo models is host-based. On the other hand, in vitro studies in a flat monoculture of tumor cells (the most frequently used in vitro tumor model) are unable to recapitulate the complexity of tumor microenvironment. Three-dimensional (3D) in vitro cell cultures of tumor cells have been proven to be an important experimental tool in understanding mechanisms of tumor growth, response to therapeutics, and transport of nutrients/drugs. We have recently described a novel tool to create 3D co-cultures of tumor cells and cells in the tumor microenvironment. Our method utilizes magnetic manipulation/levitation of the specific ratios of tumor cells and cells in the tumor microenvironment (from human or animal origin) aiding in the formation of tumor spheres with defined cellular composition and density, as quickly as within 24 h. This chapter describes the experimental protocols developed to model the 3D structure of the cancer environment using the above method.