BCR engagement initiates intracellular calcium ([Ca2+]i) mobilization which is critical for the activation of multiple transcription factors including NF-κB and NFAT. Previously, we showed that Bruton's tyrosine kinase (BTK)-deficient (btk −/−) B cells, which display a modestly reduced calcium response to BCR crosslinking, do not activate NF-κB. Here we show that BTK is also essential for the activation of NFAT following BCR engagement. Pharmacological mobilization of [Ca2+]i in BTK-deficient DT40 B cells (DT40.BTK) does not rescue BCR directed activation of NF-κB and only partially that of NFAT, suggesting existence of additional BTK-signaling pathways in this process. Therefore, we investigated a requirement for BTK in the production of diacylglycerol (DAG). We found that DT40.BTK B cells do not produce DAG in response to BCR engagement. Pharmacological inhibition of PKC isozymes and Ras revealed that the BCR-induced activation of NF-κB requires conventional PKCβ, whereas that of NFAT may involve non-conventional PKCδ and Ras pathways. Consistent with an essential role for BTK in the regulation of NFAT, B cells from btk −/− mice display defective expression of CD5, a gene under the control of NFAT. Together, these results suggest that BCR employs distinct BTK-dependent molecular mechanisms to regulate the activation of NF-κB versus NFAT.