Salivary gland adenoid cystic carcinoma (ACC) is a slow-growing but often fatal tumor due to its propensity for perineural invasion and distant metastases along nerves to the lungs and brain.  Current treatment options are limited to surgery with or without radiation; however, with a lack of targeted therapies-owing to its orphan status and limited molecular insight into its etiology- ACC continues to exhibit high levels of recurrence and metastasis.  As recent as 2009, cell lines for ACC were revealed to be contaminated or misidentified leaving the ACC field with a dearth of tools available for improving clinical outcomes of ACC.  Here, we utilized a highly efficient ROCK-inhibitor-based cell culture approach to generate several ACC cell lines and short-term cultures from ACC xenografts and primary tumors.  Confirmation of cell line identity began with microsatellite analysis of eight short tandem repeats (STRs) to validate the origin of primary cultures as derivatives of parental tumors.  As further evidence of derivation, cultures retained expression of at least some markers characteristic for ACC.  Finally, a subset of cultures exhibited tumorigenicity when injected into immunodeficient mice.  Tumors formed from injected cells recapitulated the morphology and protein expression patterns of the parental tumors.  Cell lines and derived xenografts provide an invaluable tool for the study of ACC and open new avenues for research.