Mycobacterium tuberculosis (Mtb), is the causative agent of tuberculosis (TB), a disease that still claims over 1 million lives each year. During infection, Mtb produces numerous lipid virulence factors in order to cause disease and the bacteria relies on host lipids (fatty acids and cholesterol) as nutrients that support bacterial persistence. Here, I present my research focused on understanding therapeutic weaknesses in Mtb’s lipid synthesis pathways and my studies aimed at defining the host lipid transport systems in Mtb. Mtb relies on de novo lipid synthesis to generate acyl chains needed to produce phospholipids, lipoproteins, enzyme cofactors, mycolic acids, and polyketide virulence factors. We identified a novel lead compound (sALT629) that inhibits de novo lipid biosynthesis in Mtb. This compound severely disrupts the Mtb lipidome, inhibits the incorporation of metabolic tracers into bacterial lipids, and selectively depletes triacylglycerol (TAG) levels. I also found that mutations that inactivate the hydroxyacyl-ACP-dehydratase (HadC) confer resistance to sALT629 by sustaining TAG levels in the bacterium. The excess TAG produced in a HadC mutant likely serve as a surplus of acyl chains for Mtb to mobilize when de novo lipid synthesis is blocked by sALT629. Importantly, this resistance mutation is associated with significant bacterial fitness costs suggesting that this resistance mechanism will not evolve in the clinic. To maintain persistent infections, Mtb relies on the Mce1 and Mce4 transporters to import fatty acids and cholesterol, respectively. Recent evidence indicates that these closely related transporters share protein subunits (MceG and LucA) which prevent proteolytic turnover of the transporters. Thus, we deleted MceG and identified proteins that are degraded to discover unknown subunits of the Mce1 and Mce4 transporters. Additionally, it has been reported that the protein Mce1N competes with MceG and negatively regulates Mce1 function in Mycobacterium smegmatis (Msm). Unlike in Msm, we found that Mce1N is required for fatty acid utilization and Mce1 stability in Mtb. Finally, we identified conditions where Mtb utilizes fatty acids independently of Mce1, suggesting that Mtb possesses an auxiliary fatty acid transport system. Based on these findings, we developed a screening assay that can be used to identify fatty acid transporters.