EBEX (the E and B EXperiment) is a balloon-borne telescope designed to measure the polarisation of the cosmic microwave background radiation. During a two week long duration science flight over Antarctica, EBEX will operate 768, 384 and 280 spider-web transition edge sensor (TES) bolometers at 150, 250 and 410 GHz, respectively. The 10-hour EBEX engineering flight in June 2009 over New Mexico and Arizona provided the first usage of both a large array of TES bolometers and a Superconducting QUantum Interference Device (SQUID) based multiplexed readout in a space-like environment. This successful demonstration increases the technology readiness level of these bolometers and the associated readout system for future space missions. A total of 82, 49 and 82 TES detectors were operated during the engineering flight at 150, 250 and 410 GHz. The sensors were read out with a new SQUID-based digital frequency domain multiplexed readout system that was designed to meet the low power consumption and robust autonomous operation requirements presented by a balloon experiment. Here we describe the system and the remote, automated tuning of the bolometers and SQUIDs. We compare results from tuning at float to ground, and discuss bolometer performance during flight.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.