Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis, a disease that has plagued mankind for millennia and remains problematic today. Over 1.3 million people died of tuberculosis in 2012, and many more remain latently infected with the potential to develop active TB. Although we have effective antibiotics, issues of drug resistance, lengthy treatment time, and drug side effects necessitate the development of novel therapies. Mtb, like other bacterial pathogens, depends on central carbon metabolic (CCM) pathways to generate energy and biosynthetic precursors, as well as to prevent buildup of toxic metabolites. These pathways provide a reservoir of potential drug targets, and growing evidence supports a role for CCM enzymes in growth and persistence of Mtb in vivo. However, there remains a paucity of information about the importance, function, and redundancy of specific enzymes. Here, we focused on studying the roles of two metabolic enzymes, fructose 1,6-bisphosphate aldolase (FBA) and malate synthase (MS), for Mtb growth, survival, and in vivo pathogenicity. Using conditional knockdown strains for FBA and MS, we found that both enzymes were essential for establishment and maintenance of infection within the mouse model. Interestingly, essentiality of both in vitro was dependent on the carbon source condition; FBA was essential for survival in media with single carbon sources whereas MS was essential for survival in media with acetate or fatty acids. We also observed metabolic perturbations associated with exposure of MS and FBA knockouts to death-inducing carbon sources, suggesting that death may be induced by metabolite imbalance or toxicity. Together, these data validate MS and FBA as drug targets worth pursuing and highlight the importance of recognizing that gene essentiality can be context-dependent.