Penicillium expansum, the causal agent of blue mold, exhibits a broad host range and patulin-producing capability compared with the citrus-specific pathogens P. digitatum and P. italicum. To elucidate the molecular basis underlying its successful colonization of apple fruit, we performed a comparative transcriptomic analysis of the three species during the early stages of infection. A superficial peeling inoculation method was established to improve fungal RNA recovery, yielding markedly higher genome mapping ratios than conventional wound inoculation. Cryo-SEM observations revealed rapid germination and tissue penetration by P. expansum, whereas the other two species exhibited delayed development and progressive collapse on the apple fruit. Transcriptome profiling showed distinct infection-stage-dependent regulatory patterns, with P. expansum specifically enriching pathways associated with membrane dynamics, hydrolase activity, ribosome biogenesis, and carbon metabolism. Based on clustering analysis on the expression of 422 homologous secreted protein gene pairs, sixteen highly expressed candidate virulence genes in P. expansum were selected for functional validation. Gene-knockout assays identified six secreted proteins—including a 1,3-β-glucanosyltransferase, isoamyl alcohol oxidase, amidase, pectate lyase, endoglucanase, and a predicted effector—as contributors to pathogenicity. These findings uncover new virulence factors and highlight the unique infection strategy that enables P. expansum to successfully colonize apple fruit.