Because tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) (Apo2 ligand) preferentially kills malignant cells while sparing normal cells, it may be therapeutically useful against cancers, including those of haematopoietic origin. Although the activity of TRAIL has been studied in tumour cell lines and in a limited number of different primary tumours, its overall activity in a large number of uniform cases of primary tumours is not known. We therefore studied the activity of TRAIL in 29 primary precursor B‐cell acute lymphoblastic leukaemia (ALL) samples. TRAIL was found to have a modest activity as it killed a maximum of 29% of ALL cells within 18 h compared with killing 75% of Jurkat cells. The sensitivity to TRAIL did not correlate with the pattern of TRAIL receptor expression or FLIP expression, as determined by Western blot analysis. The CD40 receptor, which can transduce survival signals in mature malignant B cells, was less frequently expressed on ALL cells, but incubation with an exogenous soluble CD40 ligand trimer did not rescue them from spontaneous apoptosis and did not mediate their resistance to TRAIL. Further, although ALL cells expressed TRAIL protein, they failed to kill target Jurkat cells in a TRAIL‐dependent manner. Our data delineate major biological differences between mature and precursor malignant B cells and suggest a limited therapeutic role for TRAIL as a single agent in primary B‐cell ALL.