Liver sinusoidal endothelial cells (LSECs) differ, both structurally and functionally, from endothelial cells (ECs) lining blood vessels of other tissues. For example, in contrast to other ECs, LSECs possess fenestrations, have low detectable levels of platelet endothelial cell adhesion molecule 1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE‐1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli—namely, extracellular matrix and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte–fibroblast) we were able to prolong the expression of both SE‐1 and phenotypic functions of LSEC such as factor VIII activity and AcLOL uptake in cocultured LSECs through the production of short‐range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE‐1 as CD32b. Conclusion: Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease. (HEPATOLOGY 2009.)