With the continuing increase in drug resistant tuberculosis (TB) seen globally, new antibiotics to treat the disease are urgently needed. A limitation to the development of effective drugs is the lack of validated drug targets. To this end we have conducted a genome wide screen of Mycobacterium tuberculosis (Mtb) to identify “in vivo essential” genes, in a murine model of latent TB infection. Our functional genomic approach has revealed a member of a two-component regulatory system, we have termed Latency Initiating Regulator-A (LirA) that is necessary for the induction of a persistent phenotype. This regulator is unique amongst dormancy regulators, in that loss of function results in unrestricted growth in vivo, as opposed to attenuation. Through transcriptional analysis we have identified genes for effector molecules we classified as either ‘virulence enhancing proteins’ (VEP) or ‘latency enhancing proteins’. Overexpression of one VEP, a member of the PE/PPE family of proteins, results in significant macrophage death following infection. We are hopeful that by revealing processes of survival and persistence during latent TB infection, we may reveal opportunities for the development of novel anti-TB chemotherapies.