<i>Objective:</i> We present the first characterization of the cytokine expression pattern of lymph node fibroblastic reticulum cells (FRC), which are the stromal cells responsible for maintaining the highly structured nodal reticular fiber framework. <i>Methods:</i> Microarray expression profiles of cultured nodal FRC and dermal fibroblasts (DF) were compared as well as their response to TNF, IL-4, IL-6 and IL-13, cytokines responsible for intranodal stromal activation. <i>Results:</i> Hierarchical clustering of FRC and DF short-term culture samples revealed genes that were differentially expressed in FRC and DF. Identified differently regulated genes were confirmed by RNase protection analysis, PCR or immunohistochemistry. At earlier culture time points, FRC showed higher levels of several chemokines, including CCL2/MCP-1, and cytokines, e.g. IL-6, whereas several genes related to the production of extracellular matrix and angiogenesis were preferentially expressed in early DF cultures. By 60 days in culture, FRC and DF showed similar expression patterns consistent with homogenization of specialized stromal subsets. FRC and DF showed nearly identical transcriptional responses to exogenous TNF stimulation. <i>Conclusions:</i> Cultured FRC showed an overall transcriptional profile similar to cultured DF, including parallel responsiveness to TNF, but with differences in the expression of chemotactic chemokines, which reflect their biological roles.