Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment. Macroautophagy is a multistep process involving: (1) signaling events that occur upstream of the molecular machinery of autophagy; (2) molecular machinery involved in the formation of the autophagosome, the initial multimembrane-bound compartment formed in the autophagic pathway; and (3) maturation of autophagosomes, which acquire acidic and degradative capacities. In this chapter we summarize what is known about the regulation of the different steps involved in autophagy, and we also discuss how macroautophagy can be manipulated using drugs or genetic approaches that affect macroautophagy signaling, and the subsequent formation and maturation of the autophagosomes. Modulating autophagy offers a promising new therapeutic approach to human diseases that involve macroautophagy.