The immune mechanisms that orchestrate protection against tuberculosis as a result of BCG vaccination are not fully understood. We used the immunomodulatory properties of fingolimod (FTY720) treatment to test whether the lung-resident memory T lymphocytes generated by BCG vaccination were sufficient to maintain immunity against challenge infection with mycobacteria (BCG). Mice were given daily fingolimod treatment, starting either immediately before s.c. BCG vaccination or during subsequent BCG i.n. challenge, to prevent LN effector and memory lymphocytes from entering the periphery either during priming or challenge, respectively. Treatment with fingolimod during vaccination reduced vaccine-mediated protection against subsequent infection. By contrast, BCG-vaccinated mice were protected when fingolimod was given during the infectious challenge, suggesting that memory lymphocytes that migrate to the lung following vaccination are sufficient for protection. Notably, the antigen-reactive IFN-gamma or multicytokine-producing CD4(+) T cells present in the lung when fingolimod was given during BCG challenge did not correlate with protection; however, expression of MHC class II on macrophages isolated from the lungs post BCG challenge was increased in the protected mice. We conclude that protection conferred by BCG vaccination is dependent on memory lymphocytes retained in the lung, although IFN-gamma production by this population is not correlated with vaccine-mediated protection.