The aim of the present study was to isolate chicken follicular dendritic cells (FDC). A combination of methods involving panning, iodixanol density gradient centrifugation, and magnetic cell separation technology made it possible to obtain functional FDC from the cecal tonsils from chickens, which had been infected with Eimeria tenella. CD45- dendritic cells were selected using the specific monoclonal antibody against chicken CD45, which is a marker for chicken leukocytes, but is not expressed on chicken FDC. Isolated FDC were characterized morphologically, phenotypically and functionally. The phenotype of the selected cells was consistent with FDC in that they expressed IgG, IgM, complement factors C3 and B, ICAM-1, and VCAM-1, but lacked cell surface markers characteristic of macrophages, T-, and B cells. Transmission electron microscopy confirmed their characteristic dendritic morphology. In addition, the identity of the FDC was further confirmed by their ability to trap chicken immune complexes (ICs) on their surface, whereas they did not trap naive antigen (ovalbumin) or ICs generated with mammalian immunoglobulins. Co-culturing allogeneic or autologous isolated FDC with B cells resulted in enhanced B cell proliferation and immunoglobulin production. The lack of MHC restriction, a functional characteristic feature of FDC, further reinforces the identity of the isolated cells as chicken FDC.