Daptomycin is a lipopeptide antimicrobial with in vitro bactericidal activity against Gram-positive bacteria that was first approved for clinical use in 2004 in the United States. Since this time, significant data have emerged regarding the use of daptomycin for the treatment of serious infections, such as bacteremia and endocarditis, caused by Gram-positive pathogens. However, there are also increasing reports of daptomycin nonsusceptibility, in Staphylococcus aureus and, in particular, Enterococcus faecium and Enterococcus faecalis. Such nonsusceptibility is largely in the context of prolonged treatment courses and infections with high bacterial burdens, but it may occur in the absence of prior daptomycin exposure. Nonsusceptibility in both S. aureus and Enterococcus is mediated by adaptations to cell wall homeostasis and membrane phospholipid metabolism. This review summarizes the data on daptomycin, including daptomycin's unique mode of action and spectrum of activity and mechanisms for nonsusceptibility in key pathogens, including S. aureus, E. faecium, and E. faecalis. The challenges faced by the clinical laboratory in obtaining accurate susceptibility results and reporting daptomycin MICs are also discussed.