Photosynthetic microalgae are currently the focus of basic and applied research due to an ever-growing interest in renewable energy resources. This review discusses the role of carbon-unit supply for the production of acetyl-CoA, a direct precursor of fatty acid biosynthesis and the primary building block of the growing acyl chains for the purpose of triacylglycerol (TAG) production in photosynthetic microalgae under stressful conditions. It underscores the importance of intraplastidic acetyl-CoA generation for storage lipid accumulation. The main focus is placed on two enzymatic steps linking the central carbon metabolism and fatty acid synthesis, namely the reactions catalyzed by the plastidic isoform of pyruvate kinase and the chloroplastic pyruvate dehydrogenase complex. Alternative routes for plastidic acetyl-CoA synthesis are also reviewed. A separate section is devoted to recent advances in functional genomics studies related to fatty acid and TAG biosynthesis.