A simple technique for organ culture of the thymus of 14-day-old mouse embryos is decribed. It allowed a characteristic time-dependent development of the immature thymus into a lymphoid thymus with large numbers of nondividing small lymphocytes. Most of these cells carried the T-lymphocyte antigenic markers Thy-1 and TL, and all expressed H-2 antigens as determined in cytotoxicity assays. They probably developed from precursor cells without detectable Thy-1 and TL. This development appeared to be dependent on the thymic microenvironment, since it also occurred in serum-free organ cultures and in cultures with medium supplemented with serum from nude mice.