In an effort to provide an experimental model for future experiments examining the effect of neuroendocrine hormones on early T cell development, it was questioned whether or not serum free media (SFM) could provide suitable growth conditions for fetal thymic organ culture (FTOC). An initial concern was that the recommended use of a 10% fetal bovine serum (FBS) containing media in FTOC may interfere with the analysis of neuroendocrine hormone effects on thymocyte development due to potential cross species reactions of bovine hormones. Defined, serum free assays will permit unequivocal analysis of hormone influence on thymocyte development in vitro. In order to determine the suitability of SFM for FTOC, we have conducted experiments to determine the cellular recovery during the peak of CD4+CD8+ thymocyte expansion in FTOC. Embryonic mouse day 14 fetal thymi were collected and cultured in Costar 6-well Transwell plates for 5 days using Stemspan H3000 SFM (StemCell Technologies, Vancouver, BC, Canada). The cellular contents of each thymic lobe were then counted by a Coulter cell counter and assayed for cellular markers by flow cytometry. The flow cytometry was performed using anti-mouse antibody labels for CD45, CD4, CD8, with gating on the CD45, in order to exclude non-bone marrow derived cells. At FTOC day 5 of culture, the organs cultured in SFM contained 1.0 × 106 cells per lobe, while the cell recovery from the organs cultured in 10% FBS media available in the lab was 3.0 × 105 cells per lobe. Flow cytometry indicated a CD4+CD8+ double positive population of 8.2 × 105 cells per lobe in SFM conditions and 1.0 × 105 cells per lobe in serum containing conditions. The experiment was repeated with similar results. Based on these data, the use of serum free condition in FTOC is permissive for thymocyte development from CD4-CD8- double negative to CD4+CD8+ double positive phenotype. These results indicate that SFM is a suitable substitute for FBS containing media in the FTOC assay. In addition, use of SFM can overcome the need to screen for FBS lots that support FTOC and minimize FBS associated variation in the assay. Future use of SFM in FTOC will allow analysis of the effects of individual neuroendocrine hormones on the development of early T cells, while preventing potential influence by hormones present in FBS containing media.