WRKY proteins represent a large family of transcription factors (TF), involved in plant development and defense. In all, 60 unique barley TF have been annotated that contain the WRKY domain; 26 of these are represented on the Barley1 GeneChip. Time-course expression profiles of these 26 HvWRKY TF were analyzed to investigate their role in mildew locus a (Mla)-mediated immunity to Blumeria graminis f. sp. hordei, causal agent of powdery mildew disease. Inoculation-responsive, Mla-specified interactions with B. graminis f. sp. hordei revealed that 12 HvWRKY were differentially expressed: 10 highly upregulated and two significantly downregulated. Barley stripe mosaic virus-induced gene silencing of HvWRKY10, HvWRKY19, and HvWRKY28 compromised resistance-gene-mediated defense to powdery mildew in genotypes harboring both Rar1-dependent and Rar1-independent Mla alleles, indicating that these WRKY TF play key roles in effector-triggered immunity. Comprehensive yeast two-hybrid analyses, however, did not reveal a direct interaction between these three nuclear-localized WRKY TF and MLA. Transient overexpression of all three WRKY TF in single cells expressing Mlo, which encodes a negative regulator of penetration resistance, significantly decreased susceptibility. Taken together, these loss- and gain-of-function studies demonstrate that HvWRKY10, HvWRKY19, and HvWRKY28 positively regulate the barley transcriptome in response to invasion by B. graminis f. sp. hordei.