Xylella fastidiosa causes Pierce's disease (PD), a serious disease in grapevines, and grapevine cultivars vary in susceptibility to X. fastidiosa in the field. The mechanism(s) by which this occurs has not been clearly elucidated. To explore possible mechanisms, X. fastidiosa cells from a PD strain were grown in pure xylem fluid of PD-susceptible grapevines, Vitis vinifera and V. labrusca, versus PD-resistant grapevines, V. champinii and V. smalliana. When grown in xylem fluid from the susceptible species, X. fastidiosa cells formed a heavier biofilm compared to those in xylem fluid from the resistant species. Differential expression of selected genes of X. fastidiosa cultured in the xylem fluids of V. vinifera and V. smalliana was analyzed using a DNA macroarray. Compared with xylem fluid of V. smalliana, xylem fluid of V. vinifera stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as rpfC, gacA, xrvA, gcvR, and cysB, and genes involved in biogenesis of pili and twitching motility, such as pilI, pilU, pilE and pilG. Increased expression of virulence genes likely contributes to the expression of PD symptom in the susceptible grapevines, whereas reduced expression of these genes may lead to limitation of symptoms in resistant grapevines.