Although the critical requirement of CD4 T cells in type I (insulin-dependent) diabetes mellitus (T1DM) has been well documented, information on the exact role(s) of CD4 T cells in T1DM development is still limited. Here, utilizing non-obese diabetic (NOD) mice deficient for CD154 (CD154-KO/NOD), we have identified a mandatory role of CD4 T cells as the functional source of CD154 in the initiation of T1DM. Without CD154, CD4 T cells were not capable of mediating help in disease development in NOD mice. In fact, full expression of CD154 on the CD4 T cells seems to be essential in the normal spontaneous development of T1DM, since no diabetes was observed in CD154(+/-) mice in which around half of CD4 T cells do not express CD154 at all, at least by the time they were 40 weeks old. It was also shown that transgenic expression of CD80 on beta cells of pancreatic islets, which is believed to provide beta cells with the ability to prime cytotoxic T lymphocytes specific for islet antigens, did not restore insulitis in CD154-KO/NOD mice. Taken collectively, these results indicated that CD4 T cells play a crucial role in T1DM as a source of CD154, and that the role of CD154 on CD4 T cells in insulitis may not be just to facilitate priming and expanding of auto-reactive CD8 T cells by activating antigen-presenting cells bearing islet antigens.