While microbiological resistance to vancomycin in Staphylococcus aureus is rare, clinical vancomycin treatment failures are common, and methicillin-resistant S. aureus (MRSA) strains isolated from patients after prolonged vancomycin treatment failure remain susceptible. Adaptive laboratory evolution was utilized to uncover mutational mechanisms associated with MRSA vancomycin resistance in a physiological medium as well as a bacteriological medium used in clinical susceptibility testing. Sequencing of resistant clones revealed shared and media-specific mutational outcomes, with an overlap in cell wall regulons (walKRyycHI, vraSRT). Evolved strains displayed similar properties to resistant clinical isolates in their genetic and phenotypic traits. Importantly, resistant phenotypes that developed in physiological media did not translate into resistance in bacteriological media. Further, a bacteriological media-specific mechanism for vancomycin resistance associated with a mutated mprF was confirmed. This study bridges the gap between the understanding of clinical and microbiological vancomycin resistance in S. aureus and expands the number of allelic variants (18 ± 4 mutations for the top 5 mutated genes) that result in vancomycin resistance phenotypes.