In order to effectively treat obesity, it must be better understood at the cellular level with respect to metabolic state and environmental stress. However, current two-dimensional (2D) in vitro cell culture methods do not represent the in vivo adipose tissue appropriately due to the absence of complex architecture and cellular signaling. Conversely, 3D in vitro cultures have been reported to have optimal results mimicking the adipose tissue in vivo. The main aim of this study was to examine the efficacy of a novel conjugate of a genetically engineered polymer, elastin-like polypeptide (ELP) and a synthetic polymer, polyethyleneimine (PEI), toward creating a 3D preadipocyte culture system. We then used this 3D culture model to study the preadipocyte differentiation and adipocyte maintenance processes when subjected to various dosages of nutritionally relevant free fatty acids with respect to total DNA and protein content, cell viability, and intracellular triglyceride accumulation. Our results showed that 3T3-L1 preadipocytes cultured on the ELP-PEI surface formed 3D spheroids within 72 h, whereas the cells cultured on unmodified tissue culture polystyrene surfaces remained in monolayer configuration. Significant statistical differences were discovered between the 3D spheroid and 2D monolayer culture with respect to the DNA and protein content, fatty acid consumption, and triglyceride accumulation, indicating differences in cellular response. Results indicated that the 3D culture may be a more sensitive modeling technique for in vitro adipocyte culture and provides a platform for future evaluation of 3D in vitro adipocyte function. Biotechnol. Bioeng. 2014;111: 174–183. © 2013 Wiley Periodicals, Inc.