Evaluation of the immunogenicity of embryonic stem cell derived differentiated cells is important for their potential application in cell replacement therapies and transplantations. Low immunogenicity or even an immune privileged status would enable their general use in allogeneic settings and therefore supply an unrestricted source. Based on conflicting data in terms of immunogenicity published for mouse and human ES-derived cells, the rat model was used to complement the knowledge in this specific area by a set of in vitro test systems using endothelial ES cell derivatives.This chapter describes the strategies and methods used to analyze immunogenicity of rat ES cell derived endothelial cells (RESC) in comparison to adult mature rat endothelial cells (EC). In a first characterization step, the endothelial nature of rat ES cell derived endothelial cells was proved by labelling with von Willebrand factor (vWF) as well as testing tube formation capacity on an extracellular matrix. The RESC can be characterized by their constitutive or cytokine-induced expression level of the Major Histocompatibility Complex (MHC) antigens class I and class II by Fluorescence Activated Cell Sorter (FACS) technology. Moreover, regulation of transcription factors involved in the IFNγ signaling pathway could be evaluated by detecting either the phosphorylation status by specific intracellular antibody staining followed by flow cytometric measurement or by analyzing the mRNA expression level by quantitative RT-PCR. By stimulating the RESC with IFNγ and coculturing with Carboxy-fluorescein diacetate succinimidyl ester (CFDA-SE)-labelled CD4(+) rat T cells, the ability of RESC to induce proliferation was analyzed by FACS technology. Allo-reactive cytotoxic T cells were generated in a mixed lymphocyte culture (MLC) with lymph node cells from two MHC-disparate rat strains and used to determine the susceptibility of RESC to lytic processes. Therefore, RESC were labelled with calcein and the release of this fluorochrome after coculture was measured. To analyze the response to humoral attacks, RESC were incubated with allo-antibody containing sera and rabbit complement and then cell damage was assessed by 7-actinomycin D (7-AAD) incorporation into the DNA using FACS analysis.