Adipocyte differentiation is a tightly regulated process which requires the sequential and organized expression of numerous genes and proteins. Phosphorylation of cytoplasmic proteins and key transcription factors represents a critical regulatory mechanism of the process leading to adipocyte maturation and modulation of associated metabolic pathways. Despite the recognition of the importance of protein phosphorylation in adipocyte biology, relatively little is known about the role of specific kinases in thermogenic (brown or beige) adipocyte differentiation and function. In this study, we demonstrate that the non-receptor protein tyrosine kinase 2 beta (PTK2B) plays a critical role in murine beige adipocyte differentiation. We observed that PTK2B protein expression is associated with beige adipocyte differentiation in cultured, immortalized, inguinal stromal vascular fraction cells. CRISPR/Cas9-mediated knock-out of Ptk2b results in non-differentiating white adipocytes, and differentiated beige adipocytes with significantly reduced thermogenic gene and protein expression, enlarged lipid droplet size, and altered mitochondrial respiration. Together, our data in a cell culture system provides evidence for a role of PTK2B in the differentiation of murine beige adipocytes.