Adipose tissue growth is essential to normal development and whole body metabolic homeostasis, but is also linked to many diseases. In particular, obesity is characterized by an excessive expansion of body fat (white adipose tissue, WAT) mass. The bulk of WAT is a loose association of white adipocytes held in a collagen matrix. The tissue is also highly vascularized. The vascular fraction contains locally resident precursor cells (preadipocytes) and endothelial cells that form the microcirculation. To date, the interactions between these cellular components in adipose tissue development remain poorly understood. As a first step, we investigated the reciprocal effects of adipocyte differentiation and endothelial cell growth using a collagen gel-based co-culture model. The co-culture consisted of human umbilical vein endothelial cells (HUVECs) and adipocytes derived from 3T3-L1 preadipocytes. We found that the co-culture synergistically enhanced the proliferation of the endothelial cells and the differentiation of adipocytes. Compared to a control culture seeded with only HUVECs, endothelial cell proliferation increased up to 6-fold. Compared to a control culture seeded with only 3T3-L1 cells, triglyceride (TG) accumulation, an important measure of differentiated adipocyte function, was increased up to 3.4-fold. The synergistic effects depended on both the total density and composition of the seeding mixture, with the largest increases observed at the highest total seeding density (2106 cells/ml collagen) and preadipocyte:HUVEC ratio (90:10) tested in this study. To determine the metabolic basis for the enhanced TG accumulation, we compared the metabolic flux profiles of adipocytes with and without HUVEC co-culture. Results of metabolic flux analysis (MFA) showed that the co-culture up-regulated the activities of several carbohydrate pathways, including glycolysis and the pentose phosphate pathway (PPP). Interestingly, the up-regulation in adipocyte metabolic activity critically depended on endothelial cell viability. When the co-culture was subjected to a hyperglycemic stress (4.5 g/L medium glucose), HUVEC proliferation was abolished, and the TG content was decreased by 60 % compared to a control culture seeded with only 3T3-L1 cells. Taken together, our results demonstrate that the adipocyte-endothelial cell co-culture model captures a number of in vivo WAT features that are absent in conventional in vitro models, including endothelial cell-stress mediated attenuation in lipogenic activity. Prospectively, the co-culture system could serve as a useful platform to develop models of metabolic diseases that directly impact on WAT function, for example type 2 diabetes.