The optimal use of transcription factors to determine B-lineage specificity in B-acute lymphoblastic leukemia/lymphoma (B-ALL) has not been fully investigated. We undertook an extensive immunohistochemical study of a panel of B-cell transcription factors in B- and T-ALL and Burkitt lymphoma to evaluate those with the best specificity and sensitivity. Tissue microarrays were constructed from 34 B-ALL, 19 T-ALL, and 30 Burkitt lymphoma samples. All 34 (100%) cases of B-ALL expressed PAX5; 32 (94%), BOB.1; 33 (97%), PU.1; 29 (85%), CD79a; 27 (79%), CD22; 2 (6%), CD20; 9 (26%), OCT-2; and 3 (9%), MUM1. Burkitt lymphoma cases were positive for PAX5 (30/30 [100%]), BOB.1 (27/30 [90%]), PU.1 (23/30 [77%]), CD79a (29/30 [97%]), CD22 (14/30 [47%]), CD20 (30/30 [100%]), OCT-2 (23/30 [77%]), and MUM1 (5/30 [17%]). T-ALLs were only positive for PU.1 (15/19 [79%]) and BOB.1 (12/19 [63%]). PAX5 demonstrated better specificity for B-lineage determination than BOB.1 and PU.1 and better sensitivity than CD79a, CD22, and CD20. These findings suggest that PAX5 has the greatest diagnostic usefulness and lineage determination in B-ALL, especially in cases with an inadequate specimen for flow cytometric analysis.